Hello Olga. I am not sure if this approach will work for this particular protein as the pI of the protein is very near the pH of the buffer in your resolving gel. Have you looked at any other papers where this chemokine receptor has been examined by PAGE-Immunoblotting? Personally, I would try to figure out why the SDS-PAGE didn't work as, at least in the presence of SDS the effects of the receptors intrinsic charge will be reduced. Is is possible that your SDS-PAGE protocol worked and your immunoblotting didn't? For a basic protein, you may want to try transfering using a CAPS-MeOH buffer (10 mM CAPS-NaOH, pH11/10% MeOH. Make up a 1M CAPS, pH 11 stock using NaOH to adjust the pH to 11, dilute the stock to 10 mM, add 10%MeOH, adjust to final volume with MilliQ-grade water).

Bruce

Hi Olga,

The stacking gel isn't needed for native gels. You may find precipitation at the top of the well during staining, but your protein should be visible in the resolving gel. Switch the electrodes, yes.

If you run the gel at 150 - 200 V, I recommend pre-chilling the running buffer and placing the tank on ice or in a cold room.

The following paper presents both electrophoretic theory and buffer recipes:

@ARTICLE{Chr-83,

AUTHOR = {A. Chrambach and T.M. Jovin},

TITLE = {Selected Buffer Systems for Moving Boundary Electrophoresis on Gels of Various p{H} Values, Presented in a Simplified Manner},

YEAR = {1983},

JOURNAL = {Electrophoresis},

volume = {4},

number = {3},

pages = {190-204},

doi = {10.1002/elps.1150040303},

}

Thank you for all your responses to my question.

Bruce Allen: I think the immunoblotting not working is a big possibility, unfortunately, I cannot tell where the problem lies since I get no signal or excessive non-specific binding of the antibody. For the CAPS transfer buffer, would I have to somehow charge the proteins in the gel with SDS so they can run towards the membrane?

Nicholas Spellmon: I switched the electrodes after adding Laemmli sample buffer without SDS for native proteins and without it. Both times my gel came out completely clear, no proteins present. Would this be caused by the protein's pI being the same as the gel's pH (pH8.8)? If so, could I make a gel without a stacking portion and that has a pH of 10 or 11 in order for my protein to migrate with the normal Tris Glycine buffer? would I also increase the buffer pH?

Steffen Pahlow: I have tried Blue native but, unfortunately, I do not amino caproic acid that some protocols calls for in order to dissolve the coomassie and to prevent aggregation of native proteins. I have used water instead, what is your opinion on that? Also, can I transfer it onto a PVDF membrane just like an SDS-PAGE gel and probe with antibodies? 

Engelbert Buxbaum: Thank you for the reference, I have started reading and it is clarifying some doubts I have had. 

Hello Olga. For a CAPS transfer, you run SDS-PAGE as you normally would. You only change the transfer buffer and, perhaps, the current/voltage during the transfer.

regards, Bruce

Olga,

Do you see precipitation on top of the well? You should see protein staining if your protein didn't migrate especially if the pI/pH match. Also, the Laemmli sample buffer should have Tris pH 6.8. Sample buffer pH should match the entering gel's pH 8.8.

You could try adjusting the buffer pH for the gel and running buffer, yes. If you increase the buffer pH, don't switch the electrodes.