Show work. Please enter final answer on line when applicable.
Number Correct: ____________ / 40 Score: ____________ / 12 (# correct x 0.3) You perform affinity chromatography on an E. coli lysate.
1. What does each SDS-PAGE lane show (i.e. elution, wash/flowthrough, contaminants, marker)? Lane M: __________ Lanes 1-2: __________ Lanes 3-5: __________ 2. What is the approximate size of the protein of interest? __________
3. If you wanted to maximize protein amount (purity is secondary), which lane(s) would you keep? __________
4. If you wanted to maximize protein purity (you dont want contaminants), which lane would you keep? __________
The SDS-PAGE gel shows non-induced lysate (Lane 2) and induced lysate (Lane 3). The induced lysate was centrifuged, and soluble (Lane 4) and insoluble (Lane 5) fractions are shown.
5. What is the approximate size of the expressed protein? __________
6. Is the protein in the soluble or insoluble cell fraction? __________
7. What technique would you use to purify this insoluble protein, prior to chromatography? __________ __________
Shown is an SDS-PAGE gel (A) and western blot (B). Lane 1 is non-induced total cell lysate, Lanes 2 and 3 contain the induced insoluble (Lane 2) and soluble (Lane 3) fractions, Lane 4 contains solubilized inclusion bodies. The fraction from Lane 4 was placed on a cation exchange column and the flowthrough (Lane 5) and eluate (Lane 6) are shown. 8. Circle the protein bands that are induced in Figure A, Lane 2. How do you know this is the protein of interest (Hint: use both figures)?
9. What is the molecular weight of the protein of interest? __________
10. Is the target protein found in the soluble or insoluble fraction? _________
11. Which would you keep from the IEX column, the flowthrough or eluate? _________ _________
12. At the pH used, what is the charge on the proteins in Lane 5? __________ Lane 6? __________
13. Draw a box around the unwanted protein band that was removed by the ion exchange column in Figure A.
Researchers analyzing a proteins oligomeric structure run an SDS-PAGE gel under very mild conditions (without heat treatment or reducing agents, and at 4 C), which is in a sense a hybrid SDS-PAGE/native gel where both molecular weight and oligomers can be observed. 14. Based on the data shown and your knowledge of heat treatment and reducing agents such as -mercaptoethanol (2-ME), draw this protein in its native conformation. Specifically draw or mention the oligomeric state, disulfide bridges, and weak bonds.
Researchers analyzing glycosylation run an 10 kDa protein on an SDS-PAGE gel, shown with markers (M), untreated protein (Lane 1), and protein treated with Endo H (Lane 2) below (arrows show band locations). The structure of this protein is shown below the gel, with arrows marking glycosylation sites.
15. What is Endo H, and what does it do? __________________________________
17. The researchers treated the protein from Lane 1 with Endo H overnight, and ran it in Lane 2. What does each band in Lane 2 represent? ____________________________________________________________________
18. Did Endo H cleave with 100% efficiency? Why or why not? ____________________________________________
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19. What is the molecular weight of: The protein (no sugars)? ____________ 10 kDa protein (not including sugars) The protein with one sugar? ____________ A single sugar? ____________
20. Based on the structure, how many total glycosylation sites are there? _______________________________ Based on the gel, how many glycosylation sites appear to be used? _______________________________ Is there a band in Lane 1 showing all glycosylation sites? _______________________________ Read the methods section below from E3 ubiquitin ligase activity of the trifunctional ARD1 (ADP-ribosylation factor domain protein 1). (Vichi, A. et. Al., 2005. Proc Natl Acad Sci U S A. Feb 8;102(6):1945-50). Fusion Protein Synthesis and Purification. Single colonies of Escherichia coli XL1 blue or BL21(DE3) gold (Stratagene) containing plasmids with inserts encoding GST-tagged ARD1 or related proteins were added to 5 ml of Luria- Bertani broth containing ampicillin at 100 g/ml. After incubation overnight at 37C with shaking, the culture was added to 500 ml of the same medium and incubated at 37C with shaking until A 600 = 0.6. Isopropyl--D-thiogalactoside was added (0.2 mM final concentration), and after incubation at 37C for 4 h, cells were sedimented by centrifugation (5,000 X g for 10 min) and stored at -20C. Recombinant proteins were purified essentially as described by Frangioni and Neel (20). Briefly, cells were dispersed in 20 ml of STE buffer (10 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, pH 8) containing lysozyme at 1mg/ml. After incubation for 1 h on ice and the addition of 1% Triton X-100, cells were sonified and incubated (1 h at room temperature, shaking) with DNase I (Roche Applied Science) at 44 units/ml. Inclusion bodies were collected by centrifugation (15,000 X g for 15 min) and dispersed in 20 ml of STE. After the addition of 1 ml of 20% Sarkosyl and intermittent mixing (vortex mixer, 5 s every 30 s) for 5-10 min,1 ml of 20% Triton X-100 was added, insoluble material was discarded after centrifugation (15,000X g for 10 min at 4C), and the clear supernatant was incubated (2 h at 4C) with 0.25 ml of reduced glutathione-Sepharose. The mixture was transferred to a column, and beads were washed three times with 10 ml of STE buffer. Bound proteins were eluted with three 0.5-ml portions of 10 mM reduced glutathione in 50 mM Tris/HCl (pH 8) and concentrated by using Microcon centrifugal filter devices (10,000 or 100,000 molecular weight cut-off; Millipore). The protein concentration was determined by the Bradford method (21). Purity assessed by silver staining after SDS/PAGE was >90%. After addition of propylene glycol (35% final concentration), protein (0.1-1 mg/ml) was stored in small portions at -20C. For ubiquitinylation assays, two different preparations of GST- or His-ARD1 protein were used.
21. Why did the authors use BL21(DE3) gold cells, and not wild type E. coli?
22. What is the name of the protein being purified in this method? ____________________________________
23. At what absorbance (OD600) did the authors induce the cells? ____________________________________
Why at this absorbance? __________________________________________________________________
24. How do you prepare the 20 ml of STE buffer the authors used to dissolve the cells (show work)?
____________ Tris ___________ NaCl __________ EDTA Method:___________________________________________
25. What is the purpose of EDTA in this buffer? __________________________________________________
26. What three different techniques did the authors use to lyse these bacterial cells? ______________________ ______________________ ______________________
27. What is DNase I? _______________________________
Why is it listed as units/ml and not mg/ml? _______________________________
28. What type of chromatography was used to purify the recombinant proteins? ______________________
What was used to elute the protein from the beads? ______________________
29. What technique did the authors use to concentrate the protein? ____________________________________
How did they determine the proteins concentration? ____________________________________
30. Why was propylene glycol added to the protein after concentration? _______________________________ The researchers from the previous method made several constructs of ARD1 to study the various domains, which are shown below, and numbered 1-6. Match the construct (1 through 6) with the appropriate lane on the SDS-PAGE gel to the right. Enter the numbers above each lane. Each protein shown is fused to GST (26 kDa, not shown). An amino acids M w , on average, is 112 Da. The numbers on top of the constructs (i.e. 1, 402, 574) indicate the amino acid number (which should allow you to calculate the approximate M w ). 31. ____ ____ ____ ____ ____ ____
32. Some lanes show a contaminant. What is the contaminant at the bottom of lanes 1 through 5? __________
What event likely produced these products? __________________________________________________
33. This paper studies ARD1s role in ubiquitination. Define ubiquitination and its function in the cell.
What are E1, E2, and E3? _____________________________________________________________________
The figure shows E1, E2, GST-ARD1 (1-574), GST, and GST-cytohesin-1 (C-1) added to proteins that can be ubiquitinated and run on a gel. The asterix denotes heat-inactivated protein. The samples were incubated with an anti-ubiquitin antibody. 34. This is a picture of (native, SDS-PAGE, western)? __________________
35. What proteins are required for ubiquitination in this experiment? ______________________________________________________________
36. Why is Lane 3 a smear? (Hint: similar to the Endo H question #16)
______________________________________________________________ 38. What was the purpose of the heat inactivation? _________________________________________________________________________________________
39. What type of ubiquitinating enzyme is ARD1 (E1, E2, E3)? _____________________________________
The researchers mixed some of the ARD1 constructs from the first figure to test their ability to facilitate ubiquitination. Using the gel to the left, they determined which domains are essential for ubiquitination. 40. Identify the minimum amino acid numbers and the name of the domain required for ubiquitination: ________________________________________