Rab11, a trafficking molecule does not come up as clear bands in western blotting but shows a lot of non specific bands. Same antibody when used for immunofluorescence shows intense staining. It is compatible with both immunoblotting and -fluorescence and also certain samples of the same cell line showed proper bands but some samples just refuse to show the band. The lysis buffer used contains 8M urea, chaps and tris and incubated overnight at 4 degrees and spun down the next day at 10,000 rpm for 45 mins. Laemmli buffer and few other lysis buffers have also been tried but still a proper band is not obtained. Any help is appreciated.
It is very possible that your antibody detects a structure of the native protein present in the slides. For western you denaturate your protein completely, so antibodies may not detect the protein, even if its present. Do you have another antibody available (which detects a different epitope)?
Probably a native epitope that is being recognised by the Ab, make sure you heat your sample 5 mins 100C and snap cool on ice before loading your gel! Otherwise I agree with Christian Praetorius.
But the same antibody earlier used to detect it in the sample and give clear bands as well.
Did u measure the protein before loading..??? try loading more protein. Probe the blot for B actin, this will atleast help you rule out Western blot technical errors.
transfer the same sample on NC and PVDF and probe with your ab, in my hands, some ab does not work at all on NC but work nicely on PVDF
Shreyas, I have tried taking different protein loads but does not help that.
Didier, I have probed the samples on PVDF. Also one sample out of many might just show a clean band whereas rest other samples do not. The samples are of different sets
Ruchi, Loading different concentrations is just one way to begin. It may not work always. In my experience, if you are trying to use an antibody for the first time, it helps. All the explanations mentioned above may be a reason its not working. There way be something in each step, right from harvesting the cells, that is preventing it from working. Hence dissecting it out and pinpointing the to the possible source of problem is the way I usually do.
Solubility of the protein may be the issue. Have you tried looking at the pellet following the spin (run lysate vs pellet). We are having a similar issue with solute carrier family proteins.
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