Task:
1. Why were BL21 gold cells used (what is in their genome), and what was added to induce its expression?
2. At what absorbance (OD600) were the cells induced and why?
3. How do you make 20 ml of STE (sodium chloride-Tris-EDTA) buffer?
4. What is the purpose of EDTA in this buffer?
5. How were the bacterial cells lysed (three ways)?
6. What is DNase I, and why is it in units/ml?
7. What type of chromatography was used to purify the recombinant proteins?
8. What was used to elute the protein from the beads?
9. What technique was used to concentrate the protein?
10. ARD1 is 64 kDa. Which cut-off mentioned would be required for this concentration?
11. How did they determine the protein’s concentration?
12. Why was propylene glycol added to the protein after concentration?
13. A purification table of an enzyme is shown. Fill in the table and answer the following questions:
a. Which two steps were best, and why?
b. Which step lost the most enzyme? Would you still keep this step? Why or why not?
c. Which step(s) would you omit, and why?
14. You are studying a DNA damage pathway, and have the following proteins in a mixture.Which type(s) of chromatography would you use to purify each of the following proteins from this sample? Include the eluting agent (such as NaCl) and pH of the buffer if relevant.
15. You purify a protein from an E. coli lysate using affinity chromatography and run fractions from the column on an SDS-PAGE.
a. What does each lane on the gel show (e.g. elution, flowthrough, marker)?
Lane M: ______________ Lanes 1-2: ______________ Lanes 3-5: ______________
b. What is the approximate size of the target protein?
c. Which fractions would you keep to maximize protein amount?
d. Which single fraction would you keep to maximize purity?
You induce a target protein’s expression in E. coli by adding IPTG to the media. The SDSPAGE gel shows non-induced (Lane 2) and induced (Lane 3) fractions. The induced cells were lysed and centrifuged. Soluble (Lane 4) and insoluble (Lane 5) fractions are shown.
a. What is the approximate size of the expressed protein?
b. Is the expressed protein in the soluble or insoluble cell fraction?
c. Is the protein in this fraction in its native state or misfolded?
d. What chemical would you add to refold this protein?
16. You wish to purify a novel protein from mammalian cells. You do not know this protein’s size or charge, and are thus unsure of which steps to take. Fortunately, an antibody for this protein is available. You have two cell lines: one that expresses the protein, and one that does not.
You run both cell lines on an SDS-PAGE (A) followed by a western blot (B) using the same gel. Lane 1 shows the control cell line, and Lane 2 the expression cell line. The cells that express the protein were homogenized, and the soluble (Lane 3) and insoluble (Lane 4) fractions are shown.
a. Which lane (3 or 4) contains the protein of interest?
b. Is it soluble or insoluble?
c. Two new proteins are visible in Lane 4. Which (the top or bottom band) is the target protein? How are you certain?
d. What is the molecular weight of the protein of interest?
e. What is the molecular weight of the expressed contaminant?
The fraction from Lane 4 was placed on a cation exchange column and the flowthrough (Lane 5) and elution (Lane 6) fractions are shown.
f. Which would you keep from IEX, the flowthrough or elution?
g. At the pH used, what is the charge on the proteins in Lane 5? What does each band in the – lane (noted by arrows) represent?
b. Is there a band showing ICAM-3 with all five sugars in CHO cells?
c. How many sugars do ICAM-3s typically have in CHO cells?
d. How many sugars do the bands in the + lane represent? Circle:
Triangle:
e. Did Endo H cleave ICAM-3 with 100% efficiency? Why or why not?
f. What are the approximate molecular weights the following?
ICAM-3: ________ ICAM-3 + one sugar: ________ A single sugar
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