back to Molecular Biology Homepage
back to main lab report keys page
[full-credit student answers, with comments]
1. (1 pt) As you know, some amino acids are neutral in charge, some are positively charged, and some are negatively charged. As a consequence, different proteins have quite different charge characteristics. They do not carry a uniform negative charge like DNA does. Nonetheless, in SDS-PAGE proteins migrate uniformly toward the positive electrode and are separated by the polyacrylamide almost exclusively on the basis of size. Explain how this works.
Despite varying charge properties, different protein molcules migrate almost exclusively by size in an SDS-PAGE gel because SDS ionic detergent binds to all of the proteins, giving them all nearly identical charge: mass ratios. A polyacrylamide gel is a semisolid matrix. The pore size of the gel is uniform, and offers more resistance to larger proteins than smaller ones. This means that smaller SDS-protein complexes will pass through the matrix with fewer obstructions, thus travelling faster than larger ones that are impeded by the gel matrix.
[The proteins are coated with SDS when they are boiled in sample buffer prior to being loaded in the gel. There is also SDS in the gel itself, and in the electrophoresis running buffer.]
2. (2 pts) We are using a technique called discontinuous SDS-PAGE, in which two gels are poured on top of each other. The first gel that the proteins enter is called the stacking gel, and the second is called the separating gel.
a. What is the purpose of the stacking gel?b. Name at least one characteristic of the stacking gel that allows it serve this function, and explain (hint: compare the recipes for the two gels, given in steps F6 and F13).
The purpose of the stacking gel is to condense the protein into a tight band before running it through the separating gel. This allws better resolution in the separating gel. The stacking effect works because the stacking gel is made up of a lower concentration of polyacrylamide than the separating gel. The protein stacks because the proteins move very quickly through the stacking gel, only to run into the separating gel. The proteins at the top of the stacking gel then have time to catch up with the proteins at the bottom.
[The other relevant difference between the stacking gel and the separating gel is pH, which affects the charge and thus the migration of glycine, a component of the electrophoresis running buffer. In the stacking gel (at pH 6.8), glycine "trails" the proteins and facilitates their stacking. At the separating gel pH of 8.8, glycine is negatively charged and thus runs at the dye front, leaving the proteins to separate out behind it.]
3. (2 pts) In the transfer technique described in part I, the proteins in the polyacrylamide gel are transferred onto a nitrocellulose membrane.
a. What drives this transfer, and how does this work?The transfer of proteins from the polyacryamide gel to the nitrocellulose membrane is carried out by placing the gel on top of the membrane and then running an electric current perpendicularly through them, with the positive electrode located beneath the membrane. This drives all of the protein out of the gel and onto the membrane [because the protein is coated with SDS and is thus negatively charged].
b. What is the purpose of moving the proteins from the gel onto the membrane?
The proteins must be transferred to the membrane in order to facilitate the binding of Strep-AP to the labeled protein. If the proteins were in the gel, the Strep-AP would have to be forced through the gel matrix in order to bind to the biotin-labeled protein. The reaction is much easier to carry out [and much more efficient] on a membrane.
4. (2 pts) Create a diagram of the streptavidin-AP detection process. Illustrate the layers and reagents involved, beginning with the biotin-labeled protein on the membrane and ending with the creation of exposed regions (bands) on the Xray film.
[This was a poorly-worded question. What I was looking for was a diagram of how the detection works--what binds to what, and how light is produced and detected. I got some very nice diagrams of the hands-on steps involved, which received full credit if they were complete and accurate. The following is more what I had in mind.]
5. (3 pts) Look at the protocol described in part M for the loading of the experimental gel next week. Draw a diagram of the results you would expect to see on the developed film if…
a. the experiment works beautifully, and demonstrates specific interaction between Ltv1-His and GST-Yar1
b. the experiment works beautifully, but demonstrates a nonspecific interaction between Ltv1-His and GST or the glutathione beads
c. the experiment works beautifully, but does not demonstrate an interaction between Ltv1-His and GST-Yar1, GST, or the glutathione beads
[NOTE 1: The data already obtained in week 12 (in the optimization of the Strep-AP procedure) demonstrated that Ltv1-His is present and detectable in the flow-through fractions from both GST and GST-Yar1 beads. "Working beautifully" does not necessarily mean that *all* of the Ltv1-His would bind to the beads--this would be really remarkable.
NOTE 2: This is a denaturing gel system. Protein samples are boiled in sample buffer (containing SDS and DTT, a reducing agent) before being loaded in the gel. SDS is also present in the gel and in the running buffer. These conditions do not allow for noncovalent interactions between proteins or between proteins and beads, so you won't see a "gel shift" in this experiment like the one you might see in a gel mobility shift assay. You'll just see the presence or absence of a band corresponding to Ltv1-His, always of the same molecular weight and thus always migrating in the same place. This does not mean that this experiment could not detect an interaction between Ltv1-His and the beads, however. If you are thinking that it does, you should go back over the experiment again.]
back to main lab report keys page
back to Molecular Biology Homepage
Created by:
bkbaxter@lclark.edu
Updated: 12 December 00