low MW bands left on gel during transfer to PVDF - (Jan/22/2007 )
My problem is transfering protein bands less than 30 KDa to PVDF membranes. I used 4-20% Tris-HCl gels for SDS-PAGE running at 150V for 1 hr 30 min and using 25 mM Tris, 192 mM Glycine, 0.1% SDS pH 8.3 as running buffer. I then equilibrate my gels as well as PVDF membranes prewetted in methanol for ~2-3 seconds in pre-chilled transfer buffer - 25 mM Tris, 192 mM Glycine, 20% Methanol, 0.05% SDS pH 8.3 at room temperature for 30 minutes. I use the BioRad transblot system, set up the cassettes submerged in transfer buffer. I transfer at 20V at 16C overnight. Although all my molecular weight marker bands transfer to the membranes, after staining the gels, I find that some of the lower MW bands do not transfer. Interestingly, this problem cropped up only recently. I would really appretiate if someone has any suggestions. Am I using too much methanol or too less SDS or are my fiber pads getting old?
Thanks in advance,
If this protocol of yours worked before and its not working now, try to make up new buffers and try it again.
We use 10% methanol and not use sds in our transfer buffer (This is what we use).