Lab 4 Answer Sheet Protein Folding and Protein Detection in the Laboratory PDF

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Laboratory 4: Protein Folding and Protein Detection in the Laboratory Answer Sheet For full credit answer all questions in complete sentences. 1. What are kinases and what do they do? Kinases are a type of enzyme that is responsible for speed up the transfer of phosphorylation groups.

2. What molecule provides the phosphate for the kinase to transfer (HINT: you’ll need to play the animation on slide 4 to find out)? The molecule that provides the phosphate for kinase to transfer is ATP

3. Describe the mutation that results in the BCR-ABL gene. What events that lead to the formation of this gene. The mutation in BCR-ABL happen when parts of chromosome 9 and 22 are switched. This happens when ATP binds to the proteins and causes the phosphate group to release the protein

4. How does the BCR-ABL protein differ from the normal ABL protein? The difference between the BCR-ABL and the ABL is that BCR-ABL is formed when chromosome 9 (ABL)and 22(BCR) trade places, this is known as translocation while ABL is usually regulated by other molecules and is found in the 9th chromosome.

5. How does the BCR-ABL protein promote the development of C M L ? BCR-ABL protein causes cell division which promotes the development of CML

6. You have been asked to design a drug to inhibit BCR-ABL in an effort to treat CML. Using your knowledge of enzyme catalyzed reactions and the BCR-ABL kinase, propose a drug development strategy to combat CML. As we know, kinase is responsible for adding phosphate groups with the help of ATP. If we were to make a drug that stopped the binding of ATP to the BCR-ABL protein, this would treat CML.

7. What is the name of the drug scientists developed to treat CML? How does it work? The name of the drug that scientist developed to treat CML is called Gleevec and this drug works by halting the binding of ATP to BCR-ABL.

8. Why might a general ATP-binding inhibitor be a bad idea? The idea of a general ATP binding inhibitor would be a bad idea because there are other kinases in the cell that would be affected by this.

9. Did Gleevec work? What evidence supports this claim? Gleevec did work and the evidence that it works is shown in the decline of white blood cells in response to Gleevec.

11. What happened to the white blood cell counts of the patients represented by the red line in slide 13, and what are the implications? The white blood cell counts went down for the first 60 days and then started to increase after the implication of this was that the Gleevec drug was not working as effectively as it was.

12. Why did some patients develop resistance? There was and additional genetic mutation that changed the structure and function of BCR-ABL which led to the BCR-ABL kinase to no longer inhibit in patients.

13. What does the mutation that converts threonine to isoleucine cause to h a p p e n ? The change from Threonine to Isoleucine constricts the shape of the ATP binding site so that ATP binds, but the Gleevec doesn’t.

14. What is a conformation change mutation? A conformation change mutation is when the BCR-ABL is forced into and active conformation which is a problem for Gleevec because it can only bind to inactive BCR-ABl protein hence rendering Gleevec ineffective.

15. What was the next step in solving the problem of Gleevec resistance (slide 16 video)? The next step was to find new chemicals that could overcome this conformation change mutation. Dasatinib was found to fit into the pocket of BCR-ABI which prevents ATP from binding

16. How long did it take researchers to develop Gleevec after the genetic mutation that causes CML was first found (slide 17 video)? It took 41 years for Gleevec to be developed because we still had to advance in Molecular biology, DNA sequencing and Chemistry.

17. Once the mutations that conferred resistance to Gleevec were identified, how long did it take for the next drug to be developed (slide 17 video)?? It took 5 years for Dasatinib to be developed after the mutations that conferred resistance to Gleevec was identified.

18. Complete the multiple choice quiz found below the animation and insert a screenshot of your completed quiz below. Please crop out any irrelevant information.

19. What does ELISA stand for? ELISA stands for Enzyme-Linked ImmunoSorbent Assay 20. What are ELISA assays used for in labs? Elisa is used to detect soluble substances like protein. It can be used to detect coronavirus through antibodies or proteins on the surface of coronavirus.

21. What are the three important limitations of an ELISA? Explain each. Limitation

Explanation

One limitation would be that the presence of antibodies doesn’t mean that the patient is sick

If the patient had the disease and later recovered they can keep producing antibodies which would result in a positive test.

Second limitation is that there could be other factors that give you a false negative.

If the patient is a poor producer of antibodies or has other substances in their blood, this could make it too low to measure or detect and result in an incorrect test.

Third limitation is that an unrelated antibody could give you a false positive if it reacts with an antigen

Being able to run tests more than once helps factor out any false positives

22. Read the information in the BACKGROUND tab and answer the questions below. What test can be used to determine whether a patient has an infectious or autoimmune disease? The test that can be used to determine if a patient has an autoimmune disease is ELISA 23. What does a positive result indicate? Positive results indicate that there are antibodies and that the person had a disease. 24. What is allowed to react with the target antigen? The watery substance that remains after removing the red and white blood cells is called the serum and a portion of this is allowed to react with the target antigen. 25. What is systemic lupus erythematosus (SLE)? SLE is a form of lupus where you’re own immune system can’t distinguish between antigens and its own body components and as a result affects different part of your body.

26. From Figure 1 (click on it), what are the four steps of an ELISA protocol? Step 1: bind sample to support Step 2: Add the primary antibody Step 3: Add a secondary antibody-enzyme conjugate Step 4: Add a substrate

27. Move forward in the simulation by clicking on the animation. In step 1, you centrifuge the samples. What does a centrifuge do? In step 1 the centrifuge is used to separate solutions into different phases (liquid or solid). This is an important step because if its skipped you may get a false positive

28. What are you preparing in step 2? Why are there three different solutions? Type your answer into the left box, and insert a screen shot of your dilutions in right box.. Please crop out any irrelevant info. We are preparing three dilutions to determine the level of antibodies. There are three different solutions to determine the concentration of a sample to find the sensitivity of the assay.

29. In steps 3 and 4, you prepare an ELISA plate. What has the ELISA plate been pretreated with? Why? The ELISA plate has been pretreated with antigens to bind SLE antigens so that the antigen would stick to the plastic of the ELISA plate and result in a formation of color from the enzyme bound to the second antibody. 30. What is the positive control? (Step 4) The positive control is the anti-DNA primary antibody. 31. What is a primary antibody? Please define. A primary antibody is the first immunoassay used to detect foreign particles. 32. What is the negative control? (Step 4) The negative control is a buffer. 33. Why is it necessary to have a positive and a negative control? (Step 4) It is necessary to have a positive and negative control so that it can rule out problems such as the change of biological and chemical reagents used in ELISA or not being conducted under appropriate conditions. One control should lead to a positive response while the other leads to a negative response. If the control sample fails to react the way it should than the sample is not trustworthy and the assay has to be done again. 34. Why incubate the plate in step 5? The plate needs to be incubated to ensure that the antibodies in the sample interact with the antigen accordingly

35. Next, in step 6, the plate is washed. Why wash the plate? The plate is washed to help remove antibodies that did not react with any SLE antigens. Any antibodies that did not react with an antigen are washed off the plate while antibodies that reacted with an antigen remain in the well 36. In step 7, a secondary antibody is added. What is a secondary antibody? Please define. !The secondary antibody is an antibody taken from another species, in this case a rabbit. The secondary antibody is added to ensure that it recognizes and reacts to the SLE antibody. If the well was not washed properly, other human antibodies will react with the secondary antibody.

37. What is the attached enzyme in this assay? (Step 7) The enzyme attached in this assay is the HRP(horseradish peroxidase) enzyme 38. What is the specific substrate for HRP? What color does it produce? (Step 7) !The specific substrate of HRP was ABTS (2,2’-azinobis-3-ethylbenzothiazoleine-6-sulfonic acid) and produces conversion of colorless substrate to a colored product, in the case yellow. 39. How can the yellow color be quantitatively measured? At what wavelength? (Step 10, in "why") By pairing of HRP and hydrogen peroxide produces a yellow solution that can be quantitatively measured at a wavelength of 414 nanometers.

40. Record your results. Indicate on this page and on the computer which boxes turned color (2 poitns) A B C + (positive) – (negative) 1:2 1:10 1:100 (P.S. i’m not quite sure what happened with the A column but that whole column is supposed to be yellow.)

41. Insert a screen shot of your results below. Please crop out any irrelevant material.

Post lab questions 1. What causes CML? CML is a type of blood cancer and is caused by the BCR-ABL protein

2. Define each of the following levels of protein structure: primary secondary tertiary and quarternary. -The primary structure is the sequence of amino acids -The secondary structure is formed by hydrogen bonds and result in an alpha helix or beta pleated sheet -The tertiary structure is the overall three dimensional shape of the polypeptide that is created from the interactions of R-groups and result in the formation of the backbone to bend and fold. Some rgroup interactions include: Hydrogen bonds, hydrophobic interactions, Van Der Waals interactions, covalent disulfide bonds, and ionic bonds Quartenary: The quaternary structure occurs when there are two or more polypeptides that bind together to form a single structure

3. What is protein coagulation? Protein coagulation is a change in structure of the protein due to heat or acids, typically from liquid to solid. A good example of this is eggs or cheese.

4. What happens when you fry an egg? When you fry an egg you are actually denaturing the egg white protein, hence the gel-like appearance.

5a.Did you complete the ELISA correctly? (Yes/No) yes, I completed ELISA correctly

Answer either b or c, depending on your answer to the previous question. 5b. If No, explain what you did wrong and what you will need to do next time. For more information, check your printable summary page. Did your incorrect procedure provide you any results? Explain what went wrong. N/A 5c. If Yes, what do the results indicate about: Patient A: Patient A is likely to have SLE Patient B: Patient B might have SLE, but more testing is required to come to a conclusion Patient C: Patient C probably does not have it....