You have completed your thesis proposal, and it is now time to make a presentation. This part, just like the rest, is vital since it will determine how soon your name will find its way to the graduation list. Here are a number of issues that one needs to consider when preparing for an oral presentation.
Time Recommendation for The Oral Presentation
Ensure that your oral defense PowerPoint covers a minimum of 20 minutes but can go as high as 40 minutes. Also, remember to reserve some time for questions and answers. Overall, your defense should take a maximum of two hours.
Usually, the student, the chair, and a second committee member will attend the oral defense. However, if your member doesn’t show up, the chair can proceed to conduct the excise. The member who misses the event will review it later and provide their vote.
As you prepare for the defense, ensure to take time and proofread the slides. Makes edits where possible. Each slide should contain the same background and font.
Notes of Your Oral Defense
Be keen not to use many colors to avoid distractions.
Before stepping into that defense room, rehearse, rehearse, and rehearse and have it at the back of your mind that time is of the essence and, therefore, should be kept. Your appearance needs to be professional and maintain a smooth flow of your content all along the presentation. Avoid the temptation of reading the slides to your audience as this might create a wrong impression to you assessors. Your slides have the sole purpose of being a guide. Where possible, employ graphics relevant to your topic to help to put your points across.
Notably, PowerPoint presentation needs to contain insignificant verbiage
It should have a maximum of five bullet points or say two brief sentences.
What Happens During Your Defense?
In case you conduct the defense on the mobile phone or PC with your committee members, forward your slides to them before presentation time.
First, you will make your presentation, and then the committee will present their questions to you for answers.
During the Oral Defense
The committee makes a vote
The presentation is accepted or accepted but needs a few revisions here and there under the chair’s supervision.
On the other hand, the presentation may be declined. In such an instance, one will be required to make significant changes subject to committee review, but no oral presentation is required.
Also, the presentation may be declined and major revisions demanded by the committee members subject to their review with additional oral presentation.
Now, it is time to delve how your proposal PowerPoint should be like
The slide (Title Page) should contain;
The degree program you are enrolled in and are of specialization
Proposal oral defense
The following slide should contain the background of your research
In this area, provide a summary of your literature research concerning the topic. Provide a vivid explanation regarding the gap in the knowledge your study will address
Expressly state the research problem. As a researcher, you ought to give evidence of consensus that indeed your problem is current and significant to the discipline. Importantly, your problem should be designed so that it either builds upon or counters the research already carried out.
Purpose of Study
Your committee members need to know whether your study is qualitative or quantitative or if it takes a mix of the two has been employed. Ensure to discuss the study’s intent and remember to provide both the independent, dependent variables and covariate variables as may be applicable.
In the slides above, you have stated the method to be applied in your research. In the design/method part, you need to tell your assessors what you chose above will help you accomplish. In this case, you will need to say such statements as “employing this method will enable me to…..”
Sample size or participants
In this part, state your intent to use either secondary data or primary data in a bid to accomplish your goal. This is the point where you will point out where you have selected your sample size from. Also, provide the criteria you used to include or exclude the participants. Additionally, give the power calculations in a quantitative study or the proposed sample size in qualitative research.
Collection of data
State the criteria you will use to collect your data, either primary or secondary data. Also, mention the software you are going to use and the instrument or survey to be used.
Analyzing Data Plan
Here, each question of the research is stated with the proposed analysis plan.
Implications for the Study (Social)
This is the stage where you give the expected implications, especially for positive social change. Bound yourself by the study scope.
Wondering what a lab report is and how one can write a good lab report?
Well, look no further!
To begin with, let us take a look at the definition of what a lab report is.
A lab report is a written document that describes and analyses a laboratory experiment that investigates a scientific concept.
And now that we have known the definition, how about we go straight into knowing how you can write a good lab report, shall we?
A good lab report entails a title, an introduction, procedure, results, discussions, and a conclusion. They are explained below.
A good lab report is supposed to draw the attention of the one reading your report. It should clearly represent the work presented.
When writing a title for a good lab report, the use of ‘the’ as the first word in the title should be avoided. This is because the use of ‘the’ may result in misleading searches when one uses the database.
A lab report should have an introduction because it gets to explain where the concepts were obtained from.
The introduction should start in a broad way and then become more specific.
Now, when writing the introduction
Start off with a very broad introduction to the topic.
Next, narrow down the introduction to talk more specifically about the topic you are investigating, and why the study you did was so important.
The introduction should also include a review that discusses what is already known about the topic. This is where you will summarize the research you have done about your topic.
Here, you should indicate what properties of the system you are measuring. Normally, the properties of the system being measured are the characteristics of the system change. I.e. Changing the temperature, and even measure its effect such as the length of a metal rod.
You should specify such measurement details as the type of standard or instrument used to make the measurement. For example, a meter ruler, vernier caliper, etc.
It is important to also give instrument uncertainties. For example, if using a meter rule, one can say that the length of the rod is measured using a laboratory meter rule accurate to within 1 cm. You may also give, if necessary, an apparatus diagram.
Additionally, when writing a lab report please take note of the following:
Describe the exact procedure you followed when carrying out your experiment.
Describe in sufficient detail to allow for the replication of findings.
Be concise in your description and omit irrelevant details. You don’t need to include details regarding instructions.
Assume the reader has no knowledge of what you did and ensure that he/she would be able to replicate your study exactly by what you write in this section.
Write in the past tense.
Do not justify or explain the method. An example is when you start explaining why you chose a particular sampling method.
Only give enough detail for someone to repeat the experiment. By this, it simply means that you are concise in your writing.
Here is where one presents the descriptive statistics followed by inferential statistics.
The results section is where one reports the means and standard deviations for each level.
In the results, one should:
Name the statistical test being used.
Report appropriate statistics.
Report the magnitude, i.e. Are the results significant or not?
Avoid interpreting the results (save this for the discussion).
Make sure the results are presented clearly and concisely. A table can be used to display descriptive statistics if this makes the data easier to understand.
Do not include any raw data.
After the results is the discussion.
Its purpose is to interpret your results, that is, explaining, analyzing, and comparing them. In simpler terms, the discussion is the point in which the researcher stands back from the results and talks about them within the broader context.
It is termed as the most important part of the report because it is where you demonstrate that you understand the experiment beyond the level of simply doing it.
This discussion section often begins by making a statement as to whether the findings in the lab experiment support or do not support the expected findings stated in the concept.
Additionally, the discussion also provides the opportunity to compare the results to the research of others.
As we continue, it is an important matter to note that one should not discuss any outcomes that are not presented in the results from the lab experiment.
Now when you reach here, the conclusion should be a single paragraph that sums up what happened in the experiment, whether your concept was accepted or rejected, and what this means. In simpler terms, the conclusion part is where you present your findings from the experiment.
This is where you demonstrate that there is something that has been learned something by stating that which you have learned. This is important because it helps one to understand the value of the lab experiment and convinces the one reading your report that the experiment was a success. It is important to then be specific, providing details of what you have learned about the theory or principle at the center of the laboratory.
In the conclusion, it is important that as you evaluate the outcome objectively, taking a candid and unbiased point of view. Suppose that the outcome is not close to what you expected. Even then, after checking your results, give reasons why you believe that outcome is not consistent with the expected. Make it plain, simple.
State the mismatch between the experimental results and the theory, and discuss the sources of the differences in terms of the errors by offering logical inferences.
Also, it is in the conclusion that you get to suggest improvements for the lab experiment.
So, by including all the above as explained, without a doubt, you will have written a good lab report that will for sure help you attain higher grades in your studies.
Full Court Press, Inc. buys slick paper in 1500 pound rolls for textbook printing. Annual demand is 1920 rolls. The cost per roll is $1000, the annual inventory carrying cost factor is 15 percent, and placing an order costs $250. (a) How many rolls should Full Court Press order at a time in order to minimize total annual cost? (b) What is the associated annual inventory carrying cost? annual ordering cost? total annual cost? (c) What is the annual item cost? (d) How long does an order last? (e) What is the time between orders?
Carol Philips is in charge of pharmaceutical suppliers at City General Hospital (CGH). During the past year, average forecasted daily demand for JY-25 bandages has been 20 cases. The forecast error standard deviation is 7.4 cases per day. The average lead time for this item is 10 days with a standard deviation of lead time of 1.8 days. The item cost is $140 per case, associated annual inventory carrying cost is 27% of the item cost, and ordering cost is $20. The Hospital has a targeted service level of 90% for these bandages. CGH is open 365 days per year. (a) What is the reorder point for the JY-25 bandages? (b) What is the total annual cost associated with Ms. Philip’s current policy of ordering 200 cases? (c) How much could be saved by ordering the EOQ amount?
Demand during a product’s lead time is 218 units with a standard deviation of 40 units. If the reorder point is 300 units, what is the service level?
Suds Company is a regional distributor for Wortman, a local microbrewery. The demand rate for the beer is 60 cases per day. Wortman makes deliveries to Suds in quantities of 140 cases per day. Suds’ ordering cost and annual carrying cost percentage are $20 per order and 40 percent, respectively. The cost of the beer is $18.45 per case with a lead time of 4 days. The company is open 310 days per year. What is the: (a) optimal order quantity? (b) annual inventory holding cost? annual ordering cost? total annual cost? (c) delivery cycle time? (d) reorder point?
Consider the historical demand for a product which appears in Table 1. Information pertaining to the demand probability distribution as well as expected excess inventory and expected shortages for various reorder points are given in the partially completed Table 2. The inventory carrying cost is computed annually at 35% of the item cost of $200 per unit. The annual demand is 5,000 units while the stockout cost is $10 per unit and the cost of placing an order is $75. (a) Determine the missing demand probabilities in Table 2. (b) Verify all of the entries in Table 2. (c) Determine the missing excess inventory and shortage quantities. (d) For a reorder point (ROP) of 50 units, what is the: Χ expected excess? Χ expected carrying cost per year? Χ expected short per cycle? Χ expected stockout cost per cycle? Χ expected stockout cost per year? Χ expected total cost (e) What should be the reorder point? (f) What is the service level for the optimal reorder point? (g) What is corresponding EOQ? Table 1: Demand Frequency Distribution Demand Frequency 40 40 45 270 50 450 55 160 60 80 Table 2: Demand Probability Distribution and Expected Excess Inventory / Shortages Reorder Point Demand Probability 40 45 50 55 60 40 0.04 0 0.20 0.40 0.60 0.80 45 1.35 2.70 4.05 50 -4.50 -2.25 0 2.25 4.50 55 0.16 -2.40 -1.60 -0.80 0 0.80 60 0.08 -1.60 -1.20 -0.80 -0.40 0 expected excess, e 0.00 0.20 5.55 10.15 expected carrying cost per year, E[CC} $0.00 $14.00 $388.50 $710.50 expected short, g 9.85 5.05 0.40 0.00 stockout cost per cycle, G $98.50 $50.50 $4.00 $0.00 expected stockout cost per year, E[SC] $3,136.94 $1,884.33 $188.68 $0.00 expected total cost $3,136.94 $1,898.33 $577.18 $710.50 2
SOCKS, Inc. uses blank DVDs in producing foreign language courseware. The company’s purchasing manager is reviewing the current policy of placing orders for 100 cases of DVDs at a time. Company records indicate that the annual demand is 10,000 cases (each case holds 50 DVDs). Accounting has estimated that it costs $20 per order. Inventory carrying cost for this item is $5.43 per case per year, based on a purchase cost of $15.50 and an annual carrying cost of 35%. The lead time is 3 working days. The firm operates 250 days per year. (a) How many tapes should SOCKS order at a time? (b) What is the associated: Χ total annual cost? Χ duration of a cycle in days? Χ reorder point? (c) How much will this save over the current policy of purchasing 100 cases at a time?
The supplier of blank DVDs for SOCKS, Inc. has approached management with an offer to make smaller deliveries in quantities of 80 cases per day. However, to cover the added shipping costs, the price of DVDs will increase by 2 cents per case. Should the offer be accepted?
The weekly demand for a product is normally distributed with mean and variance of 300 and 81, respectively. Lead time is 9 weeks. For a 99% customer service level, what is the: (a) mean and variance of demand during lead time? (b) safety stock? (c) reorder point?
Sharpe Cutter is a small company that produces specialty knives for paper cutting machinery. The annual demand for a particular type of knife is 10,000 units. The demand is uniform over the 250 working days per year. Sharpe Cutter produces this type of knife in lots and, on average, can produce 50 knives per day. The cost to set up a production run is $200 and the cost of carrying inventory for one year is $3.20 per knife. The company is open 50 weeks each year. Determine the: (a) order quantity that minimizes the total annual cost. (b) annual setup cost, annual inventory holding cost, and total annual cost. (c) production cycle time.
BOSS LTD is an owner-managed machinery manufacturer that specializes in producing small electric utility tractors for commercial use in London. England. Jack Boss the sole shareholder, started the business in 2012; his son, Andrew, is the controller.
BOSS LTD recently won a bid from a large client. To ensure that BOSS LTD can deliver this large order, the new client wants to review the audited financial statements before finalizing the contract. In particular, they have indicated that they are interested in seeing the extent of BOSS LTD revenue.
You are an associate articling with the firm of Ron & Harry Chartered Professional Accountants (RH). BOSS LTD has recently engaged RH as the auditor for the company. In prior years, BOSS LTD’s financial statements were audited by a local public accounting firm. Emma White, the partner responsible for the file, has already dealt with all client acceptance issues.
It is now October 31, 2019. You are meeting with Jennifer, who provides you with the following details.
Emma: Here are the draft September 30, 2019, financial statements (Exhibit I). I met with Jack and Andrew last week to discuss the audit; here are my meeting notes, with some general information about BOSS LTD (Exhibit II).
I would like you to prepare the audit planning memo for this new client. Please ensure that the overall financial statement risk, materiality determinations, and the preliminary analytical review of the financial statements are included. Your analysis should tie into the key accounts and assertions impacted. You do not have to provide audit procedures based on your analysis, as I will do that myself.
As inventory is BOSS LTD’s largest asset, another associate, Mark, attended the inventory count on September 30. He has documented his observations of the count and the procedures he performed (Exhibit III). I would like you to develop the follow-up audit procedures we will perform for each key inventory account assertion. As well, I would like you to review Mark’s observations of the count and identify the risks and control improvements related to inventory count procedures that we will communicate to the client.
Mark has already sent out the accounts receivable and legal confirmations. Here is a summary of the results (Exhibit IV). He did not have time to discuss the results with the client, so you will have to do all of the follow-up work. If you identify any potential adjustments to the financial statements, please prepare them, so I can add them to the summary of identified misstatements. Also, please explain what audit procedures you will perform related to the accounts receivable and legal confirmation results.
Comparative Financial Statements
As at September 30, 2019
Property, plant, & equipment
LIABILITIES AND SHAREHOLDER’S EQUITY Current liabilities:
Accounts payable and accruals
Income taxes payable
Current portion of note payable
Other liabilities (Note 1)
Note 1 – This includes contingent liabilities.
Exhibit I (continued)
Comparative Financial Statements
As at September 30, 2019
STATEMENT OF INCOME
Cost of goods sold
Advertising & promotion
Interest on notes payable
Office & supplies
Repairs & maintenance
Income before taxes
Gain(loss) on investments
Net income after tax
Exhibit I (continued)
Comparative Financial Statements
As at September 30, 2019
STATEMENT OF RETAINED EARNINGS
Net income after tax
Retained earnings, opening
Retained earnings, closing
(Prepared by Emma White, CPA)
Since its inception in 2012, BOSS LTD has had increasing income.
Peter is involved in all aspects of the business. He negotiates prices for parts with the suppliers, ensures that manufacturing of the tractors is up to standards, is involved in the hiring of employees, and reviews the financial statements.
Andrew is a CPA; he joined his father in the business in 2016, when he received his designation. Matthew receives an annual bonus of 3% of net income before tax.
This year, Boss gave all of his staff a bonus, as they had received no wage increases in the last two years due to the sluggish economy. The total bonus amount paid was £25,000.
Jack and Andrew are not concerned about implementing additional controls, as they feel that BOSS LTD’s processes are straightforward and its employees are trustworthy.
Boss sold a robotic welder for £3,000, as it was fully depreciated and was no longer required for building the tractors.
The financial processes are fairly manual; BOSS LTD uses an off-the-shelf accounting package for its GL. With the current potential growth, Jack and Andrew feel that the current GL application may no longer meet their needs.
Observation of Inventory Count and Count Procedures Performed
(Prepared by Mark, associate)
Observation of BOSS LTD’s Inventory Count on September 30, 2019
Inventory ranges from inexpensive parts, such as nuts and bolts, to valuable items, such as motors. In addition to raw materials, inventory also includes completed tractors. There is no work in process.
A manager, who deals with BOSS LTD’s inventory on a daily basis, supervised the count; most of the counters were those office staff available at the time.
There weren’t any written count procedures, but the manager said he told each count team which area to count, and asked that one member of the team perform the count and that the other member re-count it before writing it down.
Rather than marking which areas are counted, BOSS LTD uses the pre-numbered count sheets to ensure all inventory is counted.
Each count team took a pre-numbered count sheet from the manager, recorded their counts on the count sheet, and then handed it back to the manager.
When a difference was found between the count sheet and inventory records, a re-count was performed. In the cases where there was still a difference, the manager considered this an error, and investigated the difference prior to changing the inventory records.
The counters counted all inventory on the shelves, no matter what condition it was in. I asked Andrew Boss about this, and found that the only time BOSS LTD writes off inventory is when it is taken off the shelves to be used, but is found to be damaged or obsolete. According to Andrew Boss, this does not happen very often.
Shipments were received during the count, and one staff person was dedicated to receiving the shipments into the inventory system, and placing those items on the shelves.
Count Procedures Performed
I inspected the inventory for items that looked old or damaged, and found the following:
Two engines that looked old, as they were dusty
Three boxes of nuts that were rusty
10 sets of tires that were deflated
I performed test counts of the inventory. I selected a sample of 15 from the count sheets and traced them to the physical inventories on the shelf, and selected a sample of 15 from the physical inventories on the shelf and traced them to the count sheets. I found one error, in which the count sheet indicated that there were 290 gearboxes; when I counted, I found 250 gearboxes. When I informed the inventory manager of this difference, there was a re-count, and 250 was found to be the correct number. The inventory manager adjusted the amount in the system.
I obtained a copy of the final inventory listing and the count sheets.
Accounts Receivable and Legal Confirmation Results
Legal Confirmation Results:
Description of Claim
BOSS LTD’s Evaluation
Wrongful dismissal of
This lawsuit arose in
Legal counsel agreed
2017. We believe we
with BOSS LTD’s evaluation.
employee of BOSS LTD. Leon
will settle for £67,000
was fired for not
before it reaches court,
and we have accrued
protocol. He is suing
Injury lawsuit of Mary
The lawsuit arose in
The evaluation provided
2018. We believe we
by the lawyers is a
Mary was an
will settle for £48,000
settlement of £78,000.
employee of BOSS LTD, who
before it reaches court,
was injured on the job
and we have accrued
in the repair shop.
Lawsuit from Carefore
Not identified by BOSS LTD
Patni & Sons
Carefore Ltd., a
supplier of BOSS LTD, is suing
for £30,000 for breach
of contract in 2018. The
lawsuit will be settled for
£15,000 before it
Accounts Receivable Confirmation Results:
Note – We sent five confirmations to high value accounts, which make up a significant portion of total AR population.
BOSS LTD AR Sub-ledger
Invoice said we
received eight tractors,
but we received seven.
Emb Grocery Ltd.
We sent the cheque for
£40,124 on September
Halal Halal Inc.
Used a Euros exchange
rate of 0.75527.
Pata Shoes Ltd.
We received the goods
on October 2, 2019.
Ignore PST, GST & HST
Prepare an audit planning memo for the audit working paper files and include:
Assessment of overall risk at the financial statement level (pervasive risks). Include the risk factor and any associated factors that may decrease the magnitude of risk. Use the attached form to identify the risk, the accounts and transaction level assertions that may be affected and your assessment. In your memo, summarize your overall risk assessment and explain your reasoning. (hint: refer to entity-level controls covered in Chapter 7)
Calculate materiality (use the attached form). Identify the users and their needs. Determine an appropriate base and performance materiality. Summarize your conclusions on materiality in the memo.
Perform the preliminary analytical procedures based on the draft financial statements. Calculate gross profit margin (%), current ratio, a/r turnover and inventory turnover. Interpret your findings and identify any impact on the audit (additional procedures that should be performed, etc.)
Identify at least five control deficiencies in the client’s inventory count procedures along with implications and recommendations to improve. Summarize your findings in a draft memo to the client.
Identify substantive procedures to gather evidence for valuation, existence, completeness, cut-off and rights and obligations assertions related to the inventory account balance. (hint: given the control deficiencies identified, what other tests should the auditor perform to gather sufficient evidence). Summarize your findings in a separate memo to the audit engagement partner.
Analyze the legal and accounts receivable confirmations and identify proposed adjustments to account balances along with any other procedures that should be performed to gather additional information related to these confirmations. (hint: you will be drawing on your understanding of how assets and liabilities are properly measured and disclosed). Summarize your adjustments in a separate memo to the audit engagement partner.
A COB student is randomly selected, find the following probabilities (please show your work and report your probabilities as a percent rounded to the nearest percent)
The totals are from Fall 2019 enrollment as of 8/23/19 but the distribution by class standing are estimates.
All majors are disjoint.
a) The probability that this student is a sophomore: answer153/631 = .24247 or 24%
b) The probability that this student is a junior and majoring in accounting.Answer38/631 =0.0602 or 6%
c) P(Management U Senior): Answer p(management) 137/631 = 0.2171, p(senior) 160/631 = 0.2536, p(senior and management) 33/631= 0.0523 p(management or senior) 0.2171 + 0.2536 – 0.0523 = 0.4184
d) If this student is a senior, what is the probability they are majoring in economics?Answer(3/631) / (160/631) = 0.0048 or 0.5% =0.0188
e) Are class standing and major independent? Please support your answer mathematically.
f) Why did I make the caveat that all majors are disjoint? What does this mean?
g) You are interested in understanding the number of scholarships awarded to COB students. The scholarship office gives you the following information: Students can earn a maximum of 3 scholarships each year. 30% of COB students will earn 1 scholarship, 25% will earn 2 scholarships, and 20% will earn 3 scholarships. Treating number of scholarships as the random variable, construct a probability model for number of scholarships. Number of Scholarships (X) P(x=X)
h) Find the expected value and standard deviation for the number of scholarships.
i) Let us convert this into a binomial model where a success is defined as earing a scholarship and a failure is defined as not earning a scholarships. Using the information from part g, find p and q.
j) If the COB has 800 students, are the conditions met to use the normal model to approximate this binomial situation?
k) Using the normal model, what is the probability that at least 600 students earn a scholarship?
l) Using the normal model, find the IQR.
m) Using the normal model, what is the probability that no more than 620 students will earn a scholarship.
You are a CPA in a mid-size firm, Angelica Ruggle is your client and has come to you for some tax planning advice to reduce her 2019 income tax liability. Angelica is married and has a 17-year old son, Chucky who is a senior in high school and has no income. She owns Angelica’s Cafe, which she expects to produce taxable income of approximately $120,000 this year. Angelica’s 2019 taxable income is $40,000 without considering the income from the cafe. Angelica tells you that she needs to purchase new kitchen equipment that will cost $25,000. The café is very successful, and she expects its taxable income to be $170,000 in 2020. Her other taxable income is expected to remain at $40,000. Angelia’s after-tax rate of return is 10%. Write a memo to Angelica offering her two options to reduce her taxes. Include a calculation of her current tax liability before the tax savings options. For each recommendation, include a thorough calculation of her tax savings. You may also submit an Excel file to show your calculations.
For answers contact us via firstname.lastname@example.org
11- Implement the linear optimization model that you developed for Valencia Products in Problem 4 on a spreadsheet and use Solver to find an optimal solution. Interpret the Solver Answer Report, identify the binding constraints, and verify the values of the slack variables by substituting the optimal solution into the model constraints.
12.Implement the linear optimization model that you developed for ColPal Products in Problem 5 on a spreadsheet and use Solver to find an optimal solution. Interpret the Solver Answer Report, identify the binding constraints, and verify the values of the slack variables by substituting the optimal solution into the model constraints.
13.Implement the linear optimization model that you developed for Burger Office Equipment in Problem 6 on a spreadsheet and use Solver to find an optimal solution. Interpret the Solver Answer Report, identify the binding constraints, and verify the values of the slack variables by substituting the optimal solution into the model constraints.
14.Implement the linear optimization model that you developed for the investment scenario in Problem 7 on a spreadsheet and use Solver to find an optimal solution. Interpret the Solver Answer Report, identify the binding constraints, and verify the values of the slack variables by substituting the optimal solution into the model constraints.
*15.Implement the linear optimization model that you developed for Bangs Leisure Chairs in Problem 8 on a spreadsheet and use Solver to find an optimal solution.
Interpret the Solver Answer Report, identify the binding constraints, and verify the values of the slack variables by substituting the optimal solution into the model constraints.
Suppose that Mr. Bangs wants to limit the number of Adirondack chairs to at most 20. Modify and re-solve your model to determine the new solution.
Suppose that Mr. Bangs does not want to spend more than 40 hours each month on any one activity. Modify and re-solve your original model to determine the new solution.
*16.Implement the linear optimization model that you developed for the Morton Supply Company in Problem 9 on a spreadsheet and use Solver to find an optimal solution. Interpret the Solver Answer Report and identify the binding constraints.
17- Implement the linear optimization model that you developed for Malloy Milling in Problem 10 on a spreadsheet and use Solver to find an optimal solution. Interpret the Solver Answer Report and identify the binding constraints.
How Solver Works
18.For the Valencia Products model in Problem 4, graph the constraints and identify the feasible region. Then identify each of the corner points and show how increasing the objective function value identifies the optimal solution.
19.For the ColPal model in Problem 5, graph the constraints and identify the feasible region. Then identify each of the corner points and show how increasing the objective function value identifies the optimal solution.
Solve Problem 11 in Chapter 13 (Valencia Products) to ensure that the number of units produced is integer-valued. How much difference is there between the optimal integer solution objective function and the linear optimization solution objective function? Would rounding the continuous solution have provided the optimal integer solution?
2.Solve Problem 12 in Chapter 13 (ColPal Products) to ensure that the number of minutes of radio and TV ads is integer-valued. How much difference is there between the optimal integer solution objective function and the linear optimization solution objective function? Would rounding the continuous solution have provided the optimal integer solution?
*3.Solve Problem 15 in Chapter 13 (Bangs Leisure Chairs) to ensure that the number of units produced is integer-valued. How much difference is there between the optimal integer solution objective function and the linear optimization solution objective function? Would rounding the continuous solution have provided the optimal integer solution?
4.For the Brewer Services scenario described in this chapter, suppose that 11 permanent employees are hired. Find an optimal solution to minimize the number of part-time employees needed.
*5.The Gardner Theater, a community playhouse, needs to determine the lowest-cost production budget for an upcoming show. Specifically, they have to determine which set pieces to construct and which, if any, set pieces to rent from another local theater at a predetermined fee. However, the organization has only two weeks to fully construct the set before the play goes into technical rehearsals. The theater has two part-time carpenters who work up to 12 hours a week, each at $10 an hour. Additionally, the theater has a part-time scenic artist who can work 15 hours per week to paint the set and props as needed at a rate of $15 per hour. The set design requires 20 flats (walls), two hanging drops with painted scenery, and three large wooden tables (props). The number of hours required for each piece for carpentry and painting is shown below:
Flats, hanging drops, and props can also be rented at a cost of $75, $500, and $350 each, respectively. How many of each unit should be built by the theater and how many should be rented to minimize total costs?
7.Joe is an active 26-year-old male who lifts weights six days a week. His rigorous training program requires a diet that will help his body recover efficiently. He is also a graduate student who is looking to minimize the cost of consuming his favorite foods. Joe is trying to gain weight, or at least maintain his current body weight, so he is not concerned about calories. His personal trainer suggests at least 300 grams of protein, 95 grams of fat, 225 grams of carbohydrates, and no more than 110 grams of sodium per day. His favorite foods are all items that he is familiar with preparing, as shown in the table Data for Problem 7. He is willing to consume multiple servings of each food per day to meet his requirements, although he cannot eat more than one steak per day and does not want to eat more than three pulled pork sandwiches a day. He needs to consume at least two servings of broccoli and one serving of carrots per day but is willing to eat two servings of carrots if necessary. Joe likes a certain brand of nutrition bars, but he would not eat more than one. Unless previously noted, he does not want more than five servings of any one food. How many servings of each food should he have in an optimal daily diet?
8- Gales Products manufactures ribbon for thermal transfer printing, which transfers ink from a ribbon onto paper through a combination of heat and pressure. Different types of printers use different sizes of ribbons. The company has forecasted demand for seven different ribbon sizes, as shown below.
The rolls from which ribbons are cut are 900 mm in length. Scrap is valued at $0.07 per millimeter. Generate ten different cutting patterns so that each size can be cut from at least one pattern. Use your data to construct and solve an optimization model for finding the number of patterns to cut to meet demand and minimize trim loss.
10- Fuller Legal Services wants to determine how much time to allocate to four different services: business consulting, criminal work, nonprofit consulting, and wills/trusts. Mr. Fuller has determined the average hourly fees and the minimum and maximum hours (for consulting and criminal work) and cases (for wills/trusts) that he would like to spend on each. He has no shortage of demand for his services. The relevant data are shown in the table Data for Problem 10. Develop and solve an integer optimization model to maximize monthly revenue.
*11.Riesemberg Medical Devices is allocating next year’s budget among its divisions. As a result, the R&D Division needs to determine which R&D projects to fund. Each project requires various software and hardware and consulting expenses, along with internal human resources. A budget allocation of $1,300,000 has been approved, and 35 engineers are available to work on the projects. The R&D group has determined that at most one of projects 1 and 2 should be pursued, and that if project 4 is chosen, then project 2 must also be chosen. Develop a model to select the best projects within the budget.
For the Valencia Products scenario (Problems 4 and 11 in Chapter 13), use the spreadsheet model to answer the following questions by changing the parameters and re-solving the model. Answer each question independently relative to the original problem.
If the unit profit for SpeedBuster is decreased to $130, how will the optimal solution and profit change?
If the unit profit for LaserStop is increased to $210, how will the optimal solution and profit change?
If an additional 1,500 units of component A are available, can you predict how the optimal solution and profit will be affected?
If a supplier delay results in only 3,000 units of component B being available, can you predict how the optimal solution and profit will be affected? Can you explain the result?
For the ColPal Products scenario (Problems 5 and 12 in Chapter 13), use the spreadsheet model to answer the following questions by changing the parameters and re-solving the model. Answer each question independently relative to the original problem.
Suppose that the exposure for TV advertising was incorrectly estimated and should have been 875. How would the optimal solution have been affected?
Radio listening has gone down, and new marketing studies have found that the exposure has dropped to 150. How will this affect the optimal solution?
The marketing manager has increased the budget by $2,000. How will this affect the solution and total exposure?
For the Burger Office Equipment scenario (Problems 6 and 13 in Chapter 13), use the spreadsheet model to answer the following questions by changing the parameters and re-solving the model. Answer each question independently relative to the original problem.
If 25% of the pine is deemed to be cosmetically defective, how will the optimal solution be affected?
The shop supervisor is suggesting that the workforce be allowed to work an additional 50 hours at an overtime premium of $18/hour. Is this a good suggestion? Why or why not?
If the unit profit for standard desks is increased to $280, how will the optimal solution and total profit be affected?
If the unit profit of standard desks is only $190, how will the optimal solution and total profit be affected?
For the Markowitz model in Example 14.10, determine how the minimum variance and stock allocations change as the target return varies between 8% and 12% (in increments of 1%) by re-solving the model. Summarize your results in a table, and create a chart showing the relationship between the target return and the optimal portfolio variance. Explain what the results mean for an investor.
Figure 15.32 shows the Solver Sensitivity Report after solving the Crebo Manufacturing problem in Chapter 13 (Example 13.10). Using only the information in the Sensitivity Report, answer the following questions.
Explain the value of the reduced cost (−0.3)(−0.3) for the number of plugs to produce.
If the gross margin for rails is decreased to $1.05, can you predict what the optimal solution and profit will be?
Suppose that the gross margin for rivets is increased to $0.85. Can you predict what the optimal solution and profit will be?
If the gross margin for clips is reduced to $1.10, can you predict what the optimal solution and profit will be? What if the gross margin is reduced to $1.00?
Suppose that an additional 500 minutes of machine capacity is available. How will the optimal solution and profit change? What if planned maintenance reduces capacity by 300 minutes?
The protein purification in Part II is to be completed in lab, working individually.
To carry out this exercise, you must familiarize yourself with the program and the possible purification steps. Before coming to laboratory, complete Part I and review the “Written Work for Protein Purification Lab” found at the beginning of Part II.
The aims of this computer simulation are:
to familiarize you with a range of protein separation techniques
to allow you to explore how these techniques work, and to see their constraints
to allow you to devise schemes to purify proteins from a mixture, using combinations of techniques
How to use the program
Protein Purification is a Windows based program. It uses the standard menu bars and drop-down menus; these can be operated using the mouse or keyboard commands. This document assumes that you are reasonably familiar with Windows. If you have not used Windows before, ask a demonstrator for some help before you start. Throughout this document you will be given the necessary information to use each part of the program. If you follow the exercises in the order in which they are set, you will learn how to use the program as you progress.
Protein Purification has an extensive Help system including: more information about the various protein separation techniques; a list of the time costs of each of the techniques; clues about strategies that you might use; and a progress report of your current separation scheme. The Help menu is available from all parts of the program and you will also find Info buttons; press these to get instant help related to the task in hand.
When you want to finish using Protein Purification, choose Go home from the Quit menu. When you are asked if you want to store your material, answer No. (Remember that you will lose your work on the protein if you answer No, so choose Cancel to return to the program if you change your mind).
Tasks to perform using the program
The exercises are divided into two parts. Part I familiarises you with the program and allows you to explore the various protein separation techniques using a simple mixture of proteins. Once you are familiar with all of these methods, in Part II you will be asked to use combinations of separation techniques to produce a pure sample of a protein from a much more complex mixture.
The tasks in Part I require short answers to be completed on this sheet, preferably before coming to lab. You are strongly advised to read all the questions in a particular section before you begin working on the computer; this should save you time, as many of the questions may be answered from a single, carefully planned experiment. When you have completed Part I there will be a group discussion when you may be asked to present your answers to the rest of the class.
The report on your work in Part II will be completed in lab and handed in. You are strongly advised to follow the guidelines on presentation.
For Part I, complete the sections on (1) SDS-PAGE, (2) Ion-exchange chromatography, and (3) Gel filtration. Sections 5 – 7 on heat treatment, ammonium sulfate fractionation, and hydrophobic interaction chromatography may also be useful for your protein purifications in Part II, but they do not work with the protein mixture Easy3_Mixture (they do work for the more complicated mixture that you’ll be using in Part II). Isoelectric focusing (4) and affinity chromatography (8) also work, but you are not allowed to use them in your purifications!
PART I Protein Purification Techniques
1. On the title page, click on Start and select Choose a Mixture (or Start from beginning) from the drop-down menu.
2. For the exercises in Part I choose the Easy3_Mixture.
3. Another dialog box pops up; this allows you to select the protein you wish to purify from the mixture by its number. For the first task choose protein 1.
4. A box appears with information about protein 1; when you have read it and noted the relevant information click OK.
5. Throughout the exercises in Part 1 you will need to keep returning to this menu to select another of the proteins from this simple mixture. To do this, from the Quit menu choose Abandon scheme and start again; then start again from the beginning.
It is a good idea before you begin the exercises, to explore the menus and to familiarise yourself with the program in general.
1) SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis)
Introduction:SDS-PAGE is a separation technique used largely for analysis. It can be utilised to discover properties of the proteins that we wish to purify using other, more preparative, techniques. It also allows us to follow the progress of our protein purification. In preparing the protein sample for SDS-PAGE the proteins are denatured and SDS (which is negatively charged) is bound to the proteins in a constant mass ratio. The result is that all the proteins have almost a constant charge to mass ratio, and during electrophoresis will be separated solely on the basis of their size. An electric field is created across the polyacrylamide gel, and the negatively charged proteins migrate towards the anode (positive electrode). As they move through the gel, the larger molecules are retarded whereas the smaller molecules can pass more easily through the pores in the gel. The result of this sieving is that the smallest molecules would reach the anode first, and the largest last. However, the electric field is turned off before the proteins reach the end of the gel, and the mass of the proteins can be estimated from the distance travelled through the gel. To estimate the relative molecular mass (Mr) of the sample proteins, standard proteins of known Mr are run on the same gel to provide a reference. To reveal the proteins on the gel a stain, such as Coomassie blue, can be used. If the protein we are interested in has been isolated previously, a specific antibody may be available for it. If this is the case then the protein in question can be identified from the array of proteins in the mixture, using an immunoblot. Note that as the proteins are denatured prior to separation by SDS-PAGE, the Mr that can be determined is that of the individual subunits, and not of the native protein. If the protein only has one subunit then of course its Mr can be estimated directly using this technique. If a protein is composed of more than one type of subunit, then more than one spot can be seen for this protein on the gel.
It is often useful to be able to perform a 2-dimensional separation of the protein mixture. The first separation involves isoelectric focusing (IEF). IEF separates proteins on the basis of their pI (isoionic point), each protein migrates in an electric field through a pH gradient until it reaches a position where the pH of the surrounding buffer is equal to the pI of the protein (see section 4, p63 for more details). The IEF is performed in a rod gel (with no SDS present). Once the separation has been completed the rod gel is moulded to the top of a polyacrylamide slab gel containing SDS. As only small amounts of protein are involved, the SDS in the gel is sufficient to bind to the proteins as they migrate into the slab gel from the rod. Thus the second dimension is SDS-PAGE, with the proteins being separated by size.
What to do:From the PAGE menu, select 1-Dimensional PAGE.
Beware, once opened, never close the electrophoresis window from the control menu box (the square in the top left-hand corner of the window), otherwise you will not be able to perform any more electrophoresis. When you have finished a separation, click on Hide gel.
a) Perform an electrophoretic separation of the protein mixture using 1D SDS-PAGE.
(i) How many proteins appear to be present in the mixture? …………………………………………….(1 point)
(ii) Using the protein standards for reference, estimate the Mr of the subunits of each of the proteins in the mixture.
c) Use the immunoblot facility to identify each of the proteins in the mixture, and complete the table. (This will involve selecting the proteins from this simple mixture in turn – see #5 on the previous page)
estimated Mrof subunit (kD)
d) In some of the exercises which follow, you will need to use 2D SDS-PAGE to discover the effectiveness of various separation techniques. You will therefore probably find it helpful sketch the positions of the proteins on this diagram, and label which one is which.
e) (i) From the results of the 2D SDS-PAGE can you be certain that there are only three proteins present in this mixture?
Introduction: Ion-exchange chromatography separates proteins on the basis of charge using an ion-exchange resin. An ion-exchange resin consists of an insoluble matrix with charged groups covalently attached. A cation-exchange resin is negatively charged, and binds positively charged ions (cations). Similarly, a positively charged resin is called an anion-exchanger. An ion which binds weakly to the resin may be displaced, or exchanged, by an ion that binds more strongly. The degree to which a protein is retained by an ion-exchange column depends on the sign and magnitude of the protein’s net charge. The overall charge of a protein depends upon the number and type of ionisable amino acid side chain groups, and the pH of its surroundings. Each ionisable side chain group has a distinct pKa, that is the pH at which it is half dissociated. For each protein there will be a pH at which the overall number of negative charges equals the number of positive charges and so it has no net charge. This is its isoionic point (pI). If the pH is below the pI the protein, then the protein molecules are positively charged and will bind to a cation exchanger. If the pH is above the pI, then the protein is negatively charged, and so will bind to an anion exchange resin. A pH equal to the pI results in the protein molecule carrying no net charge and so it will not bind to either type of exchange resin. In selecting which type of exchange resin to use, it is important to consider the pH range over which the protein is stable (and therefore functionally active).
An ion-exchange resin is mixed with a suitable buffer, of an appropriate pH, to form a slurry. This is then poured into a chromatography column. The pH of the buffer will determine the charge on the proteins to be separated. The pH of the starting buffer should be at least one pH unit above or below the pI of the protein to be bound to the resin, to ensure adequate binding. It is also important to bear in mind the pH ranges of the ion-exchangers. A weak ion-exchanger is ionised over only a limited pH range (the term ‘weak’ does not refer to the strength of the binding of the ions to the resin). The resin in the column is washed with the starting buffer, then the protein mixture is applied. Proteins will bind or pass straight through the column, depending on their charge relative to that of the resin. Those that have been bound can be eluted by changing either the pH or the ionic strength of the eluting buffer. At low ionic strength there is minimal competition between the buffer ions and the proteins for charged groups on the ion-exchanger, and so the proteins bind strongly. As the ionic strength is increased the competition increases and so the interaction between the ion-exchanger and the proteins is reduced, causing the proteins to elute, regardless of the type of ion-exchanger used.
In this exercise you will investigate the binding of the proteins from the simple mixture, to two different ion-exchange resins, using both salt (ionic strength) and pH gradients.
What to do: Choose a pair of ion-exchange media to experiment with, either DEAE- and CM-cellulose, or Q- and S-Sepharose. (Tick the appropriate box).
I am using
As this is the first time you will have encountered a chromatographic technique in this program, take some time to investigate the menus and the options available, using the following notes to help you. It is worth doing, as many of the other separation techniques in Protein Purification are presented in a similar way
Select Ion exchange chromatography from the Separation menu. A menu pops up; choose one of the ion exchange media from the top list by clicking on it (you can change your mind by clicking on another from the list) and an elution method from the bottom list; when you are happy with your selection click on OK. Another dialog box appears; type in the pH of the equilibration buffer and click on OK. A third box appears in which you enter the values for the start and end of your chosen type of gradient (either salt or pH), then click on OK. A graph, or “chromatogram” will appear. This shows the amount of protein, as detected by UV-absorption, (left-hand y-axis) against the fraction number (x-axis), and also the gradient used (scale on right-hand y-axis).
There are a number of things you can do from this screen, (and this is the same for the other separation techniques which produce a similar type of display).
ར You can perform an enzyme assay on the fractions, to discover the location of the protein of interest. From the Fractions menu select Assay enzyme activity. A graph of enzyme activity is superimposed on the chromatogram.
ར You can perform 1D SDS-PAGE on selected fractions. From the PAGE menu select 1-Dimensional PAGE. A box appears to tell you how to select up to 15 fractions for electrophoretic analysis. When you have read this, click on OK to continue, or cancel if you have changed your mind. This is another useful way of finding your selected protein, and checking for contaminants in a chosen fraction.
ར You can perform 2D SDS-PAGE on selected fractions. From the PAGE menu select 2-Dimensional PAGE. Again a box appears to tell you how to indicate the fraction to be analysed.
ར If you want to select a certain group of fractions for use in the next step of your purification scheme, from the Fractions menu choose Pool fractions. A box appears, telling you how to use the mouse to select the fractions; you can cancel at this stage, or click on OK to continue. Slide the arrow to the first of the fractions, click the left-hand mouse button, point the arrow to the last of the fractions and click with the mouse again. Select carefully with the mouse, as you do not get the chance to undo your selection. A results box appears; click on OK to continue. (The results in this box are summarised in Progress report, available from the Help menu.)
ར If you are unhappy with the results of this separation step, you can choose to Abandon this step and continue (available in the Quit menu). (NB You do not have this option if you have already pooled your fractions.).
ར If you wish to abandon your entire separation scheme, choose Abandon scheme and start again from the Quit menu. If this option is not available, choose Abandon this step first, and then Abandon scheme on the next screen. You will be asked if you really want to do this (as it means that you lose all your data), if you choose No you will be returned to the screen you just left, if you choose Yes, the box appears saying Start from stored material?
a) Using your anion exchanger with salt elution, perform three experiments using an increasing salt gradient of 0-1 M, one at pH 5, another at pH 7 and the third at pH 8.
(i) Report your results using the table (enter fraction numbers in the form 27-38, and indicate the proteins present in each peak by their identification numbers):
(ii) At pH 5, what is the charge (positive or negative) on protein 1?……………………….
Protein 2?…………………………. Protein 3?…………………………… (1 point)
What is the charge of the column (the anion exchanger)?………………………………………………….(1 point)
Will any of the proteins bind to the column? If so, which ones?…………………………………………(1 point)
At pH 7, what is the charge on protein 1?…………….. Protein 2?…………….. Protein 3?………….. (1 point)
Will any of the proteins bind to the column, and if so, which?…………………………………………….(1 point)
At pH 8, what is the charge on protein 1?…………….. Protein 2?…………….. Protein 3?…………… (1 point)
Will any of the proteins bind to the column, and if so, which?…………………………………………….(1 point)
(iii) At pH 7, which peak (peak 1 or peak 2) represents proteins which bound to the column? ……………
Which peak represents proteins which did NOT bind to the column?………………………………….(1 point)
b) How would your results differ if you had used a cation exchanger? Be specific! (1 point per pH)
Introduction: Gel filtration separates proteins on the basis of differences in their size and shape. The technique uses a gel matrix consisting of porous beads of an inert, highly hydrated gel. The gel beads are packed into a glass or plastic column, and then equilibrated with a suitable buffer solution. The protein mixture is applied to the top of the column and then buffer is added to elute the proteins from the column. The eluate is collected at the base of the column as a series of fractions. As the proteins pass down the column they penetrate the pores of the gel beads to different extents, and so travel down the column at different rates. All proteins which exceed the maximum size of the pores will be unable to enter the beads. Therefore these proteins will only pass through the solution between the beads, and so elute from the column first, in the exclusion (or void) volume. All proteins smaller than the minimum size of the pores will equilibrate completely with the buffer inside and outside the gel beads, and so spend a proportion of their time inside the beads. These proteins will therefore move more slowly through the column and will be eluted last. These proteins elute in a volume very close to the bed (total) volume of the column. The pores in the beads are not all exactly identical in size, but span a narrow range of sizes. Proteins that have sizes very similar to the range of pore sizes will be excluded from some pores, whilst entering others. These proteins of intermediate size will therefore be partially excluded from the beads to an extent that depends on their size and shape. They will elute from the column in order of molecular mass, with the largest proteins eluting first and the smallest proteins last.
What to do: Use the various gel media available in this program to investigate the principles of gel filtration. There are three series of gel media to choose from, and each gel type is available with a range of pore sizes:
Approximate fractionation range for peptides and globular proteins (molecular mass)
1500 -30 000
4000 -150 000
Sephacryl S-200 HRa
5000 -250 000
Ultrogel AcA 54b
6000 -70 000
Ultrogel AcA 44b
12 000 -130 000
Ultrogel AcA 34b
20 000 -400 000
3000 -60 000
5 000 -100 000
60 000 -400 000
aSephadex is a registered trademark of Pharmacia-PL
b Ultrogel is a registered trademark of Pharmacia-LKB
c Bio-Gel is a registered trademark of Bio-Rad Laboratories, Inc.
From the separation menu select Gel filtration. A menu pops up; choose one of the gel media from the list by clicking on it, you can change your mind by clicking on another from the list; when you are happy with your selection click on OK. A graph, or “chromatogram” will appear. This shows the amount of protein, as detected by UV-absorption, (y-axis) against the fraction number (x-axis).
a) Using the series of Ultrogel media examine the effect of increasing the pore size of the gel on the separation of the protein mixture.
(i) Complete this table with the fraction numbers occupied by each peak (eg. 32-45): 3 points
Ultrogel AcA 54
Ultrogel AcA 44
Ultrogel AcA 34
(ii) What conclusion can you make regarding the time of elution of a particular protein, as the pore size of the gel is increased? (2 points)
The point values add up to 60 points; your score will be divided by 3 to give a possible total of 20 points.
4) Isoelectric focusing(IEF)
Introduction:Isoelectric focusing (IEF) is a method for separating molecules which differ in their charge characteristics. For IEF of proteins, the protein mixture is subjected to an electric field in an inert support medium in which a stable pH gradient has previously been generated. The inert support can be either agarose or polyacrylamide. The pH gradient is formed in this by including a mixture of low molecular mass “carrier ampholytes”. The anode (positive electrode) region is at a lower pH than the cathode (negative electrode). The pH range is chosen such that the proteins to be separated have their isoelectric points within this range. A protein which is in a pH region below its pI will be positively charged, and so will migrate towards the cathode. However, as it migrates, so the pH that the protein experiences will decrease until the protein reaches a pH which is equal to its pI. At this point it has no net charge and so migration ceases. Should the protein overshoot this point, it will enter a region of pH above its pI and so become negatively charged. It will then reverse its direction of migration and now migrate towards the anode. Therefore proteins become focused into sharp stationary bands, with each protein positioned at a point in the pH gradient corresponding to its pI. The technique is capable of extremely high resolution with proteins differing by only a single charge being resolved. It is important to avoid molecular sieving effects so that the protein separation occurs solely on the basis of charge, so the chosen support medium has pores larger than the size of the proteins being separated. IEF is mainly an analytical tool, but can be used to prepare very small amounts of pure protein. In preparative IEF, if the separation has been performed in a slab gel or in a tray of gel beads, then the bands can be cut from the gel and the proteins eluted using a buffer solution.
What to do: Select Preparative isoelectric focusingfrom the Separation menu. A box appears in which you enter the pH values for the start and end of the pH gradient, then click on OK.
In this exercise you are going to explore the effects of pH range on the separation of proteins by isoelectric focusing. Conduct your own experiments and report briefly on your findings and conclusions . Use the following suggestions to help you:
1. taking a wide range around the pI of the chosen protein
2. using a narrow range around the pI of the chosen protein
3. using a range which excludes the pI of the chosen protein
5) Heat treatment
Introduction: Protein purification procedures are usually carried out at low temperature (0-4C) since most proteins are stable at low temperatures. As the temperature increases from 0C to 37-40C their stability decreases significantly. Above 40C or so, most proteins become increasingly unstable and denature. At neutral pH, denatured proteins usually precipitate. Individual proteins differ in their heat sensitivity and so this can be used for purification purposes. The temperature stability of the desired protein is determined by trial experiment, for example by following enzyme activity as the protein mixture is incubated at different temperatures, for a set period of time. The minimum temperature at which gross inactivation occurs is noted. Once this temperature is known, less stable proteins can be preferentially inactivated by incubating the crude protein mixture at a temperature 5-10C below this value for 15 to 30 minutes. Since denaturation of all cell proteins occurs to some extent at all temperatures, and only increases with increasing temperature, the total activity of the desired enzyme usually falls to some extent after heat treatment. However, it may be a useful early step for the purification of rather more heat-stable proteins.
What to do: Use Heat denaturation (available in the Separation menu) to answer these questions:
a) Which of the proteins in this simple mixture is suitable for purification using heat treatment?
b) Explain your choice of protein for question a).
Introduction: The solubility of proteins varies according to the ionic strength, and hence according to the salt concentration, of the solution. Two distinct effects are observed. At low concentrations of salt, the solubility of the protein increases with salt concentration. This phenomenon is called “salting in”. However, as the salt concentration (ionic strength) is increased still further, the solubility of the protein begins to decrease. At sufficiently high ionic strength the protein will be almost completely precipitated from solution – an effect called “salting out”. Since proteins differ markedly in their solubilities at high ionic strength, salting-out is a very useful procedure to assist in the purification of a given protein. The commonly used salt is ammonium sulphate, as it is very water soluble and has no adverse effects upon enzyme activity. It is generally used as a saturated aqueous solution which is diluted to the required concentration, expressed as a percentage concentration of the saturated solution (a 100% solution). Before carrying out a bulk separation, a test is performed to discover the salt concentrations to use. In this preliminary test the concentration of ammonium sulphate is increased stepwise, and the precipitated protein is recovered at each stage. Each protein precipitate is dissolved individually in fresh buffer and assayed for both total protein content and the amount of the desired protein (in this case an enzyme, measured by its activity). The aim is to find an ammonium sulphate concentration which will precipitate the maximum proportion of undesired protein, whilst leaving most of the desired enzyme still in solution. The precipitated protein is then removed by centrifugation and then the ammonium sulphate concentration of the remaining solution is increased to a value that will precipitate most of the enzyme whilst leaving the maximum amount of protein contaminants still in solution. The precipitated enzyme of interest is recovered by centrifugation and dissolved in fresh buffer for the next stage of purification. This technique of ammonium sulphate fractionation is extremely useful to quickly remove large amounts of contaminant proteins, as a first step in many purification schemes. It is also often employed at later stages of purification; to concentrate protein from dilute solution following procedures such as gel filtration.
What to do: Use Ammonium sulphate fractionation (available in the Separation menu) to discover a suitable protocol for protein purification using this technique. In order to carry out preliminary tests on the protein mixture, in the first box type the chosen concentration of ammonium sulphate, click on OK, a results box appears. If you wish to go back and try another concentration click on cancel in this results box, but if you wish to continue to another step in the purification, choose whether you want to use the precipitate or the supernatant by clicking on the appropriate word, then click on OK.
a) What is the maximum concentration (to the nearest whole number) of ammonium sulphate that can be added to this protein mixture without precipitating any of protein 2?
b) What is the minimum concentration (to the nearest whole number) of ammonium sulphate that can be added to the protein mixture to precipitate all of protein 2?
c) Is it possible to obtain a pure sample of any of the proteins in this mixture using ammonium sulphate precipitation alone?
7) Hydrophobic interaction chromatography
Introduction: This technique separates proteins on the basis of their binding to and elution from a hydrophobic matrix, usually octyl- or phenyl-agarose. Binding of the proteins is often carried out at a high salt concentration to favour hydrophobic interactions. Some proteins may precipitate at this high ionic strength and so need to be removed by centrifugation prior to loading the protein mixture onto the column. Selective elution of bound proteins is then carried out by applying a decreasing salt gradient.
What to do: Explore the effects of changing the starting and final salt concentrations on the separation of the three proteins using Hydrophobic interaction chromatography(select it from the Separation menu).Choose either Phenyl- or Octyl Sepharose (use the same one for the whole of this exercise) and click on OK. Enter the values the Start and End of the salt gradient in the boxes provided, and click on OK.
I am using: (check one)
a) What are the two effects of increasing the initial salt concentration on the elution profile?
Introduction: Affinity chromatography relies on the preparation of a matrix to which the protein of interest, and preferably only this protein, will bind reversibly. The matrix is usually beaded agarose, polyacrylamide or cross-linked dextran, to which a ligand has been covalently attached. The chemical nature of the ligand is determined by the known biological specificity of the protein to be purified. In the case of an enzyme, the ligand chosen would probably be a substrate or a reversible inhibitor or activator. If it is not possible to use a ligand that is absolutely specific to the molecule of interest, it is often possible to use a group-specific ligand. The matrix bearing the ligand is packed into a column, in a buffer that will be optimal for the protein-ligand binding. For example, if the ligand is an enzyme substrate, then the buffer must contain any co-factors that are required for binding. The buffer usually has a fairly high ionic strength, to minimise non-specific binding of other proteins to the ligand. The sample is applied at the top of the column and washed through the matrix. Ideally, only the protein of interest should bind. It can then be eluted specifically by the addition of a relatively high concentration of substrate or competitive inhibitor, or, failing this, by changing the pH and/or the ionic strength to disrupt the enzyme-ligand interaction. An alternative protocol can be used if an antibody, specific to the protein of interest, is available. This procedure is applicable to all proteins irrespective of their functional activities. The antibody is covalently coupled to a suitable matrix filling the column. Only the required protein will bind to the antibody and can then be eluted by procedures which weaken the antibody-antigen interaction. Affinity chromatography is a potentially powerful technique, but it can only be used when the functional activity of the required protein is known and a suitable ligand is available, or when a suitable antibody to the protein has already been obtained. Unfortunately, in many cases neither condition is satisfied, and so other protein purification methods have to be relied upon.
What to do: Use Affinity chromatography (from the Separation menu) to discover suitable purification methods for the proteins in this mixture. You will need to experiment, as you have no way of knowing the affinity of the various monoclonal antibodies available for each protein. Select a ligand from the left hand list, and choose what you want to elute the proteins with, from the list on the right, then click on OK.
a) For protein 2, discover which monoclonal antibody and elution system gives the best separation and yield.
Suggest a two step purification process capable of separating all three of the proteins from each other. There is no single correct answer to this, we will discuss the various options available in the group discussion.
PART II. Purifying a protein from a complex mixture
WRITTEN WORK FOR PROTEIN PURIFICATION LAB
Answers to the following questions are due before you do the computer exercise. All but one can be answered by using the previous sections.
1. Since proteins are quite fragile (labile) molecules, all purification steps should take into consideration the following conditions
2. Early in purification, two low resolving steps often used are
while a low capacity but high resolution step often used late in purification
3. Traditionally, one unit of enzyme activity is defined as
4. As the enzyme is purified from a mixture of proteins we measure its purity by determining the number of units of enzyme activity per milligram of protein, this measure is the term _____________ _____________of the enzyme.
5. “Fold” purification compares the _____________________of a fraction to that of the original mix.
6. Enzyme yield is defined as:
7. We will consider our purification is probably complete when the apparently pure protein yields one spot when tested by _________________________.
8. When choosing a gel type for optimal fractionation of complex proteins the gel pore size is such that the desired protein is _____________________________________________.
9. The ph at which a protein has no net charge is its ________________________________.
At this point it (will, will not) bind to an ion exchange resin. Below this pH it will assume a __________ charge and bind to a (cation, anion) exchanger.
10. DEAE cellulose or CM cellulose are effective only in the pH range ___to____. The starting buffer should be of reasonably __________ ionic strength the affinity of proteins for the ion exchange resins (increases, decreases) as ionic strength (increases, decreases).
11. The O.D.280 is monitored in each of the eluted fractions because
Turn in the “Record of Purification” for two enzymes as well as the 2-D Page gel with the enzymes of interest circled. Be sure to identify them by number. Remember it is possible that one of your enzymes is a dimer or that the isoelectric point of your enzyme is beyond the range of pH values displayed on the gel.
The report should concisely summarise your results (yield, purity and so on) for each stage of the purification, and your conclusions. You should say why you have chosen to use particular separation techniques for a given protein. The report should also include any relevant details regarding the characterisation of the proteins in question, for example estimated Mr of the subunit. Your report should conclude with your recommendations for the way in which the project should proceed, in terms of the optimum separation technique for a given protein. You do not have to report everything you have done, but if you discover that a particular separation technique is entirely unsuitable for a given protein, you could mention this, to save time for other researchers in the future.
It is strongly recommended that you keep careful notes of everything you do while you are carrying out your investigations; remember that the program does not record everything for you. However, you should not include all that you write down in your final report. Your notes will form the equivalent of a lab note book; you would not publish the entire contents of such a book as a scientific paper!
You will be assigned2 proteins to purify; you must clearly write the number of the protein to be purified at the start of the relevant section of your report. Failure to do this will result in your assignment not being marked.
You will purifyeach of your 2 proteins from a complex protein mixture in a crude mucosal extract. In each case the protein you must purify has not been isolated before. You are aware of its enzymic activity, so you can detect its presence, but there are no specific antibodies available. You have to discover the most efficient and cost effective separation method for each of the proteins assigned to you, so you need to carry out several investigations using different combinations of separation techniques to discover the optimal method. You are aiming to get a pure sample of your protein, with a high yield. The budget allocated to your project is restricted, so you must pay careful attention to the cost of each step in the purification. Your research director will monitor the progress of your project, both in terms of time and financial costs, and will intervene if it appears that you cannot meet the time and budget targets.
The proteins I have been assigned to purify from the crude extract are:
What to do: BeginProtein Purification. On the title page, click on Start and select Choose a mixture from the drop-down menu. Choose the Default Mixture and type in the number of your assigned protein.
A common strategy is to run a separation, pool your samples, and then run a 2D electrophoresis experiment to evaluate your results. A quirk of this program is that you then have to select “hide gel” from the menu before you can continue.
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1) to become sufficient in basic Excel functionality, including formulas and formatting
2) to analyze the operations of a hospitality company
In addition this project will address the following learning objectives required of this course.
University Undergraduate Learning Objectives:
-Inquiry and Critical Thinking: Graduates are able to identify problems, articulate questions, and use various forms of research and reasoning to guide the collection, analysis, and use of information related to those problems.
-Communication: Graduates are able to write and speak effectively to both general and specialized audiences, create effective visuals that support written or spoken communication, and use electronic media common to one’s field or profession.
Hospitality College Learning Objectives:
-Communicate effectively in written, spoken, visual and digital modes to different audiences (e.g. industry leaders, employees, employers, faculty and peers).
-Manage all forms of capital (e.g., human, financial) in an ethical and sustainable way.
-Analyze financial, marketing, and operational results and outcomes for hospitality operations.
You will prepare the income statement and balance sheet for 2 calendar years (2018-2019), conduct a horizontal and vertical analysis, and calculate ratios of the company you choose. As a group you will analyze these financials statements and ratios to evaluate your company performance between the 2 years.
This project is intended to integrate the material learned in class and specifically Chapters 2, 3, and 5. This project will give students an application example that is very common in industry. This project is typically the first steps hospitality management takes in not only analyzing their financial performance and making adjustments if necessary, but also preparing to conduct their forecast and budgets, which we cover in Chapter 9 and 10 (and which you will do in Project 2).
Submissions are expected to resemble those presented to an upper level manager in a hospitality operation and will give students an opportunity to apply written communication skills.
This project is to be completed using a publicly traded hospitality company’s financial statements (10-Ks). Each group must choose a different hospitality company.
Vertical and Horizontal Analysis
You are to start by building in Excel the year end 2019 and 2018 Income Statement and Balance Sheet for your hospitality firm based off the company’s 10-K (If there is a 10-KA must use that). All statements should have account names first than the 2019 numbers in the second column and 2018 in the third column. No other years will be accepted without prior approval. If your company does not do a calendar year end (or within 1 month of the year end) you must email me what their year-end is and get approval.
The Income Statement must have subtotals for Total Revenue, Total Operating Expenses, Operating Income, Income Before Taxes, and Net Income at a minimum. You may have more subtotals based on the company’s financial statements but you MUST have these 5 at a minimum. You must include all subtotals off the 10-K in addition to these stated. All subtotals must be formulas and not hardcoded numbers. If you believe your company financials do not have these subtotals you must check with the professor for a variation to this requirement. Anything turned in without these will be penalized points. If you do not know what a subtotal should include do not ignore it, come to office hours to ask.
Only go to Net Income or Net Loss. If your company has multiple Net Income/(Loss) lines due to subsidiary companies and such, go all the way to the last one right before earning per share. Do not include any Earnings Per Share information or additional information below that.
The Balance Sheet must have subtotals and totals for Current Assets, Long-term Assets, Other Assets, Total Assets, Current Liabilities, Long-term Liabilities, Total Liabilities, Total Stockholders’ Equity, and Total Liabilities and Stockholders’ Equity at a minimum. More subtotals may be added based on the company’s financial statements, but you MUST have these 9 even if the company does not show these subtotals. You must include all subtotals off the 10-K. All subtotals and totals must be formulas and not hardcoded numbers.
You are to do a horizontal and vertical analysis for each statement. You are to do all vertical and horizontal analysis possible given that you only have 2 years of data. Do not do any more years.
Calculate the following ratios for both years:
Current Ratio (coverage ratio)
Working Capital (show to nearest dollar, ie. $10,000)
Debt-to-Equity (coverage ratio)
Asset Turnover (turnover ratio)
Profit Margin (percentage ratio)
Return on Assets (percentage ratio)
Return on Equity (percentage ratio)
Earnings Per Share (Per unit basis – show in dollars and cents, ie. $1.25)
If your company is not publicly traded, you must check with the professor to get an alternative ratio to calculate. You cannot just ignore this ratio.
Solvency Ratio (coverage ratio)
Number of Times Interest Earned (coverage ratio) – If you company does not have interest expense you must see the professor to validate and be given an alternative ratio to calculate. If you calculate this and have no interest and did not check you will receive no credit on this ratio.
For any formulas that use Net Income, you must use your company’s bottom Net Income (the last one). Using a different Net Income line will cause you to get no points.
Some of these ratios may require you to get additional information that is not on the financial statements. Do not ignore those ratios, make sure to get the additional information.
All ratios must be linked to the Income Statement and/or Balance Sheet and the only numbers allowed to be hardcoded are those that do not appear on the financial statements.
Based on the horizontal and vertical analysis and the ratios you calculated, as a group select 5 accounts per statement that as management of this company you would want to analyze. You must select accounts not subtotals or totals. You should have a write-up on 10 accounts (5 from the Income Statement and 5 from the Balance Sheet). Each account must answer the following 2 questions (See Criteria section for an example). You cannot select Accounts Receivable (or an equivalent account) as an account to analyze since the example given below is on that account.
For each account you must provide a short explanation (1-2 sentences) of why you choose to analyze this account. Your reasoning for account selection should be based on class discussions, what you have learned in class, and the results of your horizontal and vertical analysis or ratio calculations. Specifically discuss what on the horizontal and vertical analysis or ratios calculations made you choose this account and this includes stating the exact numbers that caused you to select these accounts. If you choose an account that had minimal change based on your analysis you must support why you choose to look into an account that did not have a big change.
After discussing why you selected the account you must explain what caused the change in the account. You must get this information from the 10-K directly and cite the page number you got your information from. If you cannot find a valid explanation for the change you must select another account (although most accounts have a reason but it may not be explicitly stated). If you do not understand the change, please see the professor for assistance. Do not guess. Make sure you include the correct reason. For instance if the account went down (like in the example below for accounts receivable) you cannot state that the allowance for doubtful accounts, which is a contra asset account went down. This account going down would cause Accounts Receivable to go up. Also make sure to address an issue that was a main reason for the change. For instance, if the account changed $10 million, do not include an explanation for $1 million of the change when there is also information on $4 million of the change.
Team Member Evaluation
If you want (not required though) you may do a team member evaluation and turn it in on the same day you turn your project in. Just like working in teams in the real world, sometimes certain members of a group do not do their share of work even though everyone gets the same credit. After reviewing the evaluations if the majority of team members give significantly lower scores and is supported with comments and proof such as unanswered emails, that team member’s score will be lowered and if the member did no work they will receive a “0”. The evaluations along with all supporting proof should be turned in on the same date and can be turned in during office hours instead of in the classroom. Evaluations turned in with no proof will not be accepted. The instructor may request a meeting with you about your evaluation before adjusting teammates grades.
The project is due on Wednesday, October 14, 2020. Projects received after this date will be penalized 10 points (out of 100) each 24-hour period they are late (beginning at the start of class). This includes not submitting both parts online by the due date. You can earn extra credit by turning the project in at the beginning of class or before Wednesday, October 7, 2020. All projects turned in for extra credit will earn 1% point additional on your final class grade.
Projects must be submitted online (regular due date and extra credit date). No projects will be accepted for extra credit after the start of class on the due date, including even one minute late. Any projects not completed are not eligible for extra credit including not having all pages or parts attached.
It is imperative you turn in professional work and not leave this until the last minute. I will not accept unprofessional work just as your manager would not. Unprofessional work is not college level, it’s extremely hard to grade, and the scores are extremely low. If I determine your work is unprofessional, such as no formatting on the statements, multiple pages per statement, papers not straight or unreadable, etc. you will incur a 15 point penalty and be informed via email that you then have ONLY 24 hours to fix and resubmit.
All vertical and horizontal analysis and ratios must be completed using Excel. All write-ups must be completed in Word. Handwritten assignments or portion thereof are unacceptable.
You must have a vertical and horizontal analysis for the Income Statement and Balance Sheet. All statements (4 in total) and ratios should be printed “portrait” style and be on one sheet of paper each. If you cannot fit them on one sheet of paper you must check with the professor beforehand to get approval for multiple pages or points will be taken off. To get approval please bring in your copy and a copy of the 10-K statement. (Note: in case of remote learning, you will not need to submit hardcopy).
You must do this in Excel and I will be reviewing all electronic submissions to confirm you did all required formulas in Excel. Not doing this project in Excel, not submitting this online, or not using the Excel formulas will result in no credit for this part and hence a 60% point penalty on the entire project grade.
On the horizontal and vertical analysis, all dollar figures should be presented exactly like the 10-K and all percentage columns should be shown as percentages with 1 decimal (i.e. 7.2%). Make sure that your all statements are formatted with dollar signs, commas, borders, underlining, etc. and are presentable. Also make sure to note what value your company’s dollar values are in, for instance thousands or millions.
On the ratios, dollar figures should be rounded to the nearest dollar or cents depending on ratio, coverage and turnover ratios should be presented to 2 decimals (i.e. 1.12 times or 1.12 to 1), and percentage ratios should be shown as percentages with 2 decimal (i.e. 7.25%). Make sure that all ratios are formatted correctly and are presentable.
The write-ups must also be portrait style and must be double spaced in 12-point font.
You must submit your Excel submission and your Word submission to WebCampus and check your plagiarism score. Both are due before the due date and time online in addition to being turned in in class.
Spelling and grammar count. Remember you are presenting to upper management and the report should be written with that in mind. Run spell check in both Excel and Word. It is suggested you see the Writing Center if your writing skills are lacking. Points will be deducted for papers that are hard to read and if they are too hard to read it will be a “0”.
You must submit hard copies (or word file, if the class is remote) of each part of the project and you must present your work as follows in order to receive credit:
Individual Excel Analyses and Ratios
Cover Page with your name and the name of your company
Income Statement horizontal analysis
Income Statement vertical analysis
Balance Sheet horizontal analysis
Balance Sheet vertical analysis
Print out of the Income Statement from the company’s 10-K for all years used
Print out of the Balance Sheet from the company’s 10-K for all years used
Cover Page with all students’ names listed, the name of your company, and the originality score your received when you uploaded the write-ups. The originality score can be handwritten.
Income Statement write-up
Balance Sheet write-up
Print out all pages from the 10-K that you cite in your write-up. Put them in order of how you cite and highlight the area you cited. Also make sure each is numbered and in order.
This should be presented as a “unified” group project. This means that all sheets must be in the same font, format, etc. Do not just copy and paste each group member’s part into the document and submit without making sure it is consistent in format, tone, wording, etc. Remember this is a group project so you should not use “I” and instead use “we”.
Staple all of your analysis together. No binders or paperclips are allowed.
The grading rubric below should be carefully reviewed and followed.
The write-up should be no longer than ¼ page for each account and really only needs to be 2-3 sentences to address the questions sufficiently. Here is an example of a write-up that would get full credit.
As management in Boyd Gaming, we choose to look into Accounts Receivable, net. We choose this account because it decreased $38.33 million and 58.5% from 2013 to 2014. This account also decreased from 1.1% of total assets in 2013 to 0.6% of total assets in 2014. This account decreased mainly due to the allowance for doubtful accounts, which lowers the amount of accounts receivable, decreasing $19.8 million due to the company deconsolidating Borgata in Atlantic City on September 30, 2014, which caused Boyd Gaming to no longer record those assets, liabilities, and non-controlling interests in the property (Pg. 65). This one decrease accounts for over half the change, 51.7%.
You will notice both the vertical and horizontal reasons were given for selecting the account. That is not required. You can select based only on 1 of those (or the ratio calculations) if you want, but I wanted to give you both scenarios so you can see how they should be written.
This is a high-level guide to make sure the project is completed and follows all the requirements. This has been added since I have found that most points off are because students do not read all the instructions and make up their own “rules”. This is just a guide, so you don’t miss any minimum requirements.
These items are points that come right off the starting score of 100%:
Points off if not followed
-15 and required to fix within 24 hours
No Cover page missing any required information
Up to -10
Not Turning in on time including turning in on WebCampus
-10 per 24 hours
Project in not in required order as listed in #9 of Requirements
Project is not stapled (binders and paperclips not allowed) (if your class is remote, you will submit the project as a file)
10-K statements not included (any missing is total points off)
Errors in formatting, grammar, spelling, etc. (dollar signs, spelling, presentable, borders, consistency, etc.)
Up to -20
Spelling and grammar issues, after first 2
-1 point each error
These are the maximum points for each required part:
Points off if not followed
Horizontal and Vertical analysis for each statement – 10 points each
Subtotal or totals must be formulas – 2 points off for each that are hardcoded numbers even if the horizontal and vertical analyses are done correctly
Horizontal or vertical formula are correct – 5 points off each wrong one
Ratio Calculations – 2 points each calculation (1 each year)
Write Up – 4 points per account
Why you selected this account – 2 points What caused the change – 2 points
Choosing a subtotal instead of an account will have the full points taken off even if you answered all questions
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