peptide expression - (Sep/15/2005 )
i was trying to express a small peptide (40aa). since i just wanted to see whether the peptide is expressed or not. i decided to use dot blot instead of running a gel. basically here is what i did: i transfected the constructs i made into cos1 cells for 48 hours; then i extracted protein and dropped 100 ug whole cell lysate onto NC membrane; waited till the membrane to dry and block with 5% milk; primary antibody for overnight and secondary antibody for 2 hours. unfortunately i didn't see anything after developping the films. by the way, the positive control for the antibody was working, so antibody was fine. has anybody done this dot blot thing before? any suggestion is appreciated. thanks in advance.
In our lab we have spent a lot of time trying to establish the dot-blot estimation of the protein production level, but IMHO, that did not correspond to that we had in Western. IMHO, dot blot is a waste of time, nothing can replace good Western Blot, or at least, good Coomasie stained SDSPAGE gel
that depends. is the protein expressed intracellularly? if it is secreted, you may have lost it during the extraction? I am guessing you are using all your protease inhibitors and such?
Have you tried running two aliquots of the sample side by side with both western and dot blot? this would tell you if there is a problem with the dot protocol. it looks like you're doing it right?
do you soak your membrane in transfer buffer prior to dotting? most of the time you don't need to do so, but if it works with western and not with dot, perhaps it has to do with the pI of your protein or something like that, and it doesnt' want to bind the dry membrane due to pH issues
Another possibility is that, the peptide was so small, that it passed through the holes in NC film, so you have not detected them. You can try 0.2um PVDF membrane. If you run a gel, may be you should use Tris-TRicine buffer system to run the gel. It is said that as small as 3kd of a protein could be seen with a sharp band in gel run in this buffer. Finally, there is another likelihood, antigenic property depends on the complex structure of the protein, so, may be your peptide is short, so the antigencity is poor, and it is not effective to use a immunological approach to test its expression. Good luck with your experiments!.