Using the program “Protein Purification”, you will examine the efficacy of a series of gel filtration

media in separating the 20 proteins in the Default Mixture. You will be assigned to investigate EITHER
the Ultrogel series (Ultrogel AcA 54, 44, and 34) OR the Bio-Gel series (Bio-Gel P-60, P-150, and
P-300) by your instructor.

Instructions for work in lab:

1. Go to “Protein Purification”:

http://www.agbooth.com/pp_ajax/

2. Select the “Default_Mixture”, then select protein 1 (or any of the proteins; you will not be purifying
them, so it does not matter which you select). Next select your first assigned gel filtration medium from
the “Separation” menu.

3. Take two screenshots of the resulting separation (the elution profile): one should include your entire
screen (with the day and time visible)*, while the other should include only the elution profile itself
(with the name of the gel filtration medium visible). Put both screenshots into a Word document; all
screenshots that you take during lab should go into this Word document (which you should submit
before leaving lab to the site in “Lab Information” entitled “Submit Screenshots from “Protein
Purification III” During Lab (10/5 - 10/9)”)
NB: Bio-Gel P-300 calls itself “Sephadex G-50” on its elution profile! (However, it does not look
anything at all like the actual Sephadex G-50 elution profile).
*You cannot get credit for your “Results” section (see below) without these shots.

4. Now run 1-Dimensional PAGE on every 5th fraction that contains protein. For example, if fractions
20 through 95 contain protein, select fractions 20, 25, 30, 35, etc. up to fraction 95. You can select up to
15 fractions to run on a gel, so you may have to run more than one gel to cover all your protein-
containing fractions. (You may skip a fraction if it obviously does not contain any protein).

5. Take two screenshots of each gel (one of the whole screen with the day and time visible; the other of
only the gel itself) and include them in your Word document.

6. From the “Quit” menu select “abandon scheme and start again”.

7. Go back to step 2 and repeat, this time using your next assigned gel filtration medium.

Instructions for your “Gel Filtration” lab report:

Objective (2 points): include the goal of the separations you did.

Results (6 points): The screenshots of the elution profiles and 1D PAGE gels for all three of your
assigned gel filtration media (artfully arranged!) will constitute the “Results” section. Include only the
screenshots that concentrate on the elution profiles and 1D PAGE gels in this section; the whole-screen
screenshots should go at the end of your report (see below).
Formatting tip: when lining up two gels from the same gel filtration medium, remember that by using
the “Arrange” menu you can send one of them to the back or bring one of them forward!

Discussion (7 points): consider your Objective while writing the Discussion!

Some questions/issues to consider as you evaluate each gel filtration medium:

1. The void volume (elution of the largest proteins): Which peak in the elution profile represents proteins
that eluted in the void volume? Give fraction numbers.

On the basis of the 1D PAGE gels for each gel filtration medium, what do you believe is the maximum
size of the proteins which can fit into any of the pores? Explain your reasoning. (This will give you an
estimate of the exclusion size for that gel filtration medium which you can then check against the value
given by the program under the “Help” menu).

2. Elution of the smallest proteins: at the very end of the elution, were there any proteins that were not
being separated? If so, what was the range of sizes for these proteins? Explain why these proteins are not
separating.

3.According to your PAGE gels, for what size range of proteins did each column do the best job of
separation? Explain why.

NB: You should NOT answer these questions as a list in your Discussion! Instead, systematically work
your way through the elution profiles and 1D PAGE results for each gel filtration medium and explain
which proteins it can separate, which proteins it cannot separate, and WHY.

How many of the 20 proteins do you think consist of a single polypeptide (that is, are monomers)?
Why?

Whole-screen screenshots that include the day and time: place these at the end of your report. They
MUST be here, or your Results section will receive no credit.