about transfer low molecular weight protein in tricine PAGE - (Sep/11/2008 )
HELLO guys
my protein is predicted about 3kd, so i use tricine gel to run the western, but after transfer i cant see the protein that i want
anyone can give some advice. about the transfer conditon or something else, thank you very much@@
by len
Which kind of tricine gel do you use, which pore size or is it gradient? Have you made a ponceau-staining to check the transferred proteins?
use bio-rad 16.5 tris-tricine gel. 0.2 microM PVDF.
I only use amido black to check. but i cant see anything in low molecular weight area.
we dont have the ponceau-staining. thanks a lot
do you know if your antibody is working? If you don't know for sure, do you have a positive control that you can test? If you don't have a positive control, you may want to try making one - make a fusion construct with GFP or some other tag that you can easily identify visually by western and/or on a microscope. This could help you to sort out if it's the antibody that's not working or the western conditions.
Perhaps it would be better to use gradient gels 10-20%. I also had problems with western blots from Bio-Rad gels, they would not transfer well, it was really frustrating. Try to use Invitrogen-gels, they have also tricine gradient, work much better for WB. Amido-staining is also fine, just to check if the protein is there. You can also run a prestained marker which has a band somewhere near your target protein size.
what were your transfer conditions?
you may have blown the peptide through your membrane. you can test by placing another membrane behind the first during transfer and stain that one, as well.
for very small proteins and peptides you don't need to transfer for long periods.
you may also be able to find and try pvdf with 0.1 or 0.05um pores.