Hi Hoda,

maybe your problem arising from the fact that the GPCRs are membrane proteins, they can tend to aggregate when they are concentrated at the end of the stacking gel. You do not cook the samples in the sample bufffer, which is clever, because some membrane proteins can aggregate during cooking. There some ways to counter this problem, the easiest, lower the protein amount and maybe do increase the LDS concentration. Additionally try a sample buffer with LDS (or SDS) and 8 M Urea (which then is quite denaturing) and last but not least try fresh LDS (or SDS) and DTT.

Good luck,

Carsten

From the picture, the topmost band in your blank, control and transfected lanes is clearly nonspecific. Moreover, in your transfected lanes there are laddery bands, making it difficult to point out the specific band. Its the problem with your primary HA antibody. You need to re-standarize it to avoid non-specificity. Antibody against small tags like HA  or FLAG does create problems like these sometimes. You can try using a antibody against another tag fused to your protein (if present) or use antibody against the endogenous protein.

When working with membran proteins I incubate them in Laemmli sample buffer at 65°C for 5min. before loading on gel. In the loadin buffer I have an concentration of 40mM DTT.

Try to run your samples without heating or boiling. Simply mix your samples with  the sample buffer, incubate at room temperature for 10-15min,  vortex, and spin briefly and load your samples.......

Given that you get the same result with an empty vector, would it be possible to use a differennt cell line? Consider testing in HEK-293T cells, as these give high transfection efficiencies and will allow you to troubleshoot your protocol.

I would recommend that as a test, you use a reliable anti-GPCR antibody. While you will also detect any endogenously expressed GPCR, the cells in which you have overexpressed your GPCR should show a higher level. Doing this may demonstrate whether your transfection worked or not.

Best

Dear Hoda,

In addition to Ricardo's comments above, I would put some attention to the transfection method used in this particular cell type since even the transfection with empty vectors gave you smeared lines. Knowing which transfection method and cell type did you use would be of help to analyse the outcome of this experiment? Would it be possible that your transfection method is triggering a cellular response that may account for the smearing? Are you mock-transfecting your controls as well?

Cheers,

Thanks to everyone who contributed with their helpful answers. I tried using another HA-antibody and it gave much clearer bands. I mock transfected my cells to see how the transfection influences the cells. The vector was not empty though, which explains why I saw some bands. 

It is worth running the samples without heating them.  You will see sharper bands and no smearing....

Thanks Kamil :) I'll def. try.