Name: ___________________________ Student ID:__________________ Section:________

Protein Purification – Pre-Class Exercise

To prepare you for this section of the class, you will need to:
- Read Chapter 3, Section 3.3 – “Working with Proteins” from the textbook.
- Read the “Lecture Notes on Protein Purification”

You are expected to complete this worksheet by Monday at 10am and Submit Online. We will spend some time
in class answering questions over these topics before we switch to the next topic. Thus, this is your best
opportunity to see what questions you have on this topic.

You have a mixture of proteins. Among them is an enzyme you want to purify. You take your mixture and run a
2D-GEL.
a) What are the 2 axis represented in a 2D-GEL?
What properties of proteins do they take into
account for the separation?

The circle shows you your enzyme of interest. This

is the compound you are trying to purify.

b) What is the approximate MW of your enzyme?

c) What is the approximate pI of your enzyme?

With this information, you start to design your own purification protocol.

Method Protein (mg) Enzyme Specific Enrich Percent

(Units) /Total Activity Recovery /
Activity Yield (%)
Start 511 8400
Ammonium Sulfate

d) You decide to start with an Ammonium Sulfate precipitation. What is the principle behind this method?
Since there is still a lot you do not know about your protein, you decide to do a 50% saturation precipitation.
Your precipitate: 79.1% of the enzyme and 45.8% of the protein.

e) Will you keep the precipitate or the supernatant? Explain and complete the table with the appropriate
values.

f) Where would you expect to find your protein of interest if you had done the same procedure at 95%
saturation? And most of the other proteins? Explain.

With the fraction you selected you decide to do a gel filtration. Here are your options for gels.

g) If you compare Sephadex G-50 vs Bio-Gel P-300, which one do you think may do a better job of separating
your protein? Why?
You choose the Sephadex G-100 column and obtain the following
plot of the absorbance of the different fractions. The red dashed
curve indicates fractions where enzymatic activity was observed.
h) Explain why there is no protein signal during the first 5 fractions
(hint: look at the gel above).

i) Explain why most of your enzyme was found in the early fractions
and not the late fractions.

j) Which fractions would you keep and pull before the next step? Why?

You now have 78.4 mg of protein and 6641.2 of enzymatic units. Add this to your table above.

k) You now will do an ion exchange chromatography. Which of the following two matrices would you choose
and why? You will start with your column elution solution at pH7.
CM – cellulose
DEAE – cellulose

You decide to do a DEAE-cellulose column (this may or may not have been a good idea).
l) What range of pHs would your elution solution have? Explain your choice.
Turns out that you decided to do this experiment twice, each time with a different pH range (5-7 in the left
plot or 7-9 shown on the right plot) in the elution solution. You obtain the following 2 plots. Again, the red
dashed lines show enzymatic activity present.

m) Based on your understanding of ion-exchange chromatography and DEAE, explain why you only see your
enzyme eluting in the late fractions of the pH5-pH7 range DEAE elution.

You pull the fractions containing the enzyme (from the experiment on the left) and obtain the following
amounts: 33.2mg of protein and 6641.2 units of enzymatic activity.

To decide if you should continue purifying your protein, you run another 2D gel and see the following dots.
Again the circle shows you your protein of interest. Seems like you are almost there! Now we need to further
separate the 2 dots.

n) Looking at your gel-chromatography options, you can tell

that none of them can help. Why is this?

o) Same is true for ionic exchange columns. Why?

p) So what would you do? Why?

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