Question

Design a purification scheme to separate all of the peptides blow, using at least two of the methods we discussed in class sequentially. Assume you start with a homogenous solution containing all of the peptides in a buffer at pH 7.0. Explain in complete sentences WHAT you plan to do and HOW it will work to separate the peptides. Include which methods you will use and in what order, as well as the order in which the peptides will elute. You may want to include a diagram in addition to your description. (8 points)

1. DVE
2. LDQDAEEDNG

3. RLK

4. EVIEED
5. GFQVAIMGTP

6. KRKNHQ
7. VKWKIHAKSR

8. FSL
9. FLMFLA

Answer 1

All the peptides can be seperated by running a 2 dimentional gel electrophoresis. The first step involves running the mixture of peptides on a IEF( iso-electric focusing ) strip, similar to SDS-PAGE, the proteins or peptides will be pushed through the acrylamide gel by an electric field. Where IEF gets more exciting is that the gel incorporates a pH gradient, and each protein moves only until it reaches its isoelectric point (pI). The pI is the pH where a protein has no net charge, meaning the field has no effect and the protein stays put, focusing tightly into a band within 0.01 pH unit of its pI.

This strip can then be placed on a SDS page and allowed to run in order to seperate peptides of similar isoelectric point but having different molecular weight, i.e. seperation of second dimension is based on molecular weight while the first dimension is based on the isoelectric point.