Topic 12 Protein Purification Flashcards
Approximately how many types of protein does a typical cell contain? A. 50 B. 500 C. 5,000 D. 50,000 E. 500,000 F. 5,000,000
C. 5,000
What is the range of protein concentrations in cells? A. 1 ng.mL-1 to 1 µg.mL-1 B. 1 µg.mL-1 to 10 mg.mL-1 C. 1 ng.mL-1 to 100 mg.mL-1
C. 1 ng.mL-1 to 100 mg.mL-1
If you had a choice, what type of cell or tissue would you choose as your starting material to purify a protein from? A. One with a high concentration of the protein. B. One with a low concentration of the protein.
A. One with a high
concentration of the
protein.
What units should we measure ADH activity in? A. µmol. NADH B. µmol. NADH. L-1 C. µmol. NADH per second D. Elephants
C. µmol. NADH per second
Imagine we have two proteins of the same molecular
weight: gel filtration won’t separate them. But Protein A has a large proportion of lysines and arginines and Protein B has a large proportion of aspartates and glutamates.
Suppose the column is packed with media that has a permanent positive charge.
Which protein will bind to the media? A or B?
A. Protein A will bind to the media
B. Protein B will bind to the media
B. Protein B will bind to the media
Suppose we use the same positively-charged chromatography media.
But we now have a protein that has many histidines, all with a pKa of 6.0.
Will the protein bind to the media at pH 5.0?
A. Yes, at pH 5.0 the protein will
bind to the media
B. No, at pH 5.0 the protein will
NOT bind to the media
C. Some will, some won’t.
B. No, at pH 5.0 the protein will
NOT bind to the media
What charge does the protein have at a
pH greater than its isoelectric point ?
A. A positive charge
B. A negative charge
B. A negative charge
Suppose you only care about one protein in a mixture of many
proteins, is it better to bind the protein to the column or let it flow
through the column?
Scenario A: the protein of interest does not bind to the column, it
flows straight through.
Scenario B: the protein of interest binds to the column.
A. Scenario A is better
B. Scenario B is better
C. It doesn’t matter
B. Scenario B is better
Source of proteins before molecular biology
purified from tissues or cells
Sources of protein after molecular biology
Overexpress genes native to one species in
- prokaryotes
- unicellular eukaryotes
- mammalian cells
Advantages of proteins from prokaryotes
- easy to genetically manipulate
- easy to grow in large quantities
- potential high yield
Disadvantages of proteins from prokaryotes
- different post-translational modifications
2. poor at folding complicated proteins
Advantages of proteins from unicellular eukaryotes
- Easy to genetically manipulate
- Easy to grow in large quantities
- Potential high yield
- More similar post-translational modifications
Disadvantage of proteins from unicellular eukaryotes
- moderate ability to fold complicated proteins
Advantages of proteins from mammalian cells
- full range of post-translational modifications
2. folding of complex proteins
