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Chapters 7-12 Homework Name: _____________________________ Lab Section: _______ Seat: _______

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? __________________________________

____________________________________________________________________

16. Lane 1 lacks Endo H. What does each band in Lane 1 represent?

____________________________________________________________________

____________________________________________________________________

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?
____________________________________________

____________________________________________

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)

______________________________________________________________

37. Why were GST and C-1 added to Lanes 1 and 2?

______________________________________________________________
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:
________________________________________

What do C34A and H53A show?

_________________________________________________________________
1

2

3

4

5

6