small protein blotting - (Sep/22/2009 )
I'll blot a small protein (<10 kDa). I'm using nitrocellulose membrane and I'm going to do a tank-blotting, I wonder what is the voltage or current I should use to trasnfer the protein. For my condition, people told me to run it (minigel, 12mmx14mm) overnight at 100mA, but I'm not feeling quite safe to do like that since the protein is so small.
And can I load protein without boiling so it won't get degraded?
I suggest using PVDF, the nitrocellulose is porous to small proteins so you can easily blot through it if you are not careful. PVDF will hold on to the proteins.
Thanks. But I don't have PVDF at hand and our nitrocellulose membrane has a .45 microl pore size. I'll give it a try anyway - this is what I can do now. I blotted o/n yesterday at 75mA and it seems the 6kDa marker is still there, but that maybe because of the huge amount of it.
I guess you'll just have to play around with the conditions to optimize the transfer. Running it at 4 ˚C will help.
FYI 0.45 micron filters won't even filter out many bacteria - a 5 kDa protein is many millions of times smaller than this.
we use 0.2um pore nitrocellulose for small proteins and peptides (usually transferred from a tris-tricine gel) and it works fine.
0.45um pore allows too much protein to pass through, especially if you use a high current density during transfer (the protein will move too fast to be captured by the membrane and will not be significantly retarded by the pore).
Thanks! I'll change the membrane next time when I do it. But I still wonder why the 6kDa marker band is visible in ponceau stainning after transfer?
there's probably a lot more marker protein than your protein of interest, so a detectable amount gets captured by the membrane.
I also have some problem to transfer/blot low molecular weight proteins (ranging from 4kDa to 17kDa)
I use Tricine gel 10-20% and 0.22um PVDF membrane and transfer on wet ON at 15V constant but only have my 17kDa transferred.
Does anyone has transfer conditions for such proteins, I can't get the complete infos on this topic: Wet vs. semi dry, Voltage, time of transfer...etc.
Thanks a lot for your help,
i think you are transferring too long. check the recommended conditions in the manual that came with your apparatus.