no detection of proteins at 25KDa - (May/11/2011 )
Suddenly got this problem. Was doing transfer of proteins (ranging from 110-20KDa) at 35V Overnight, 10% gels. Im using PVDF membranes and transfer buffer has tris, glycine and methanol at the standard concentrations. Im looking at proteins at 25KDa. I ran the gel with marker, and probed with the antibodies I had probed earlier with (primary 2 hours, secondary 1 hour at RT, blocking 5% non-fat milk, 0.5% tween)in gels which I transferred at 100V, 1hour (12.5%)Strangely this time I could not detect anything on both blots. However, this time blocking of the membranes went for 3 hours (usually I do it for 1-2 hours). The marker has transferred perfectly. I would probe these membranes with other antibodies just to see if anything went wrong with the whole blot!! I was sure I did not mess up with the poles when I put the transfer...
Thanks in advance
did all of the markers transfer perfectly (including the low mw markers)?
the poles are correct if the markers transferred well.
the lower mw proteins may have blown through the membrane, you may have over-transferred.
what is the porosity of your membrane? 0.2um is better for low mw than 0.45um.
why so much tween? most use 0.05-0.1%.
Thanks for the reply...apologise for the typo...tween is at 0.05% (as always)...the lower molecular weight markers transferred perfectly (till 10Kda, I can see them on the membrane). Actually, I went ahead to look for some proteins at 60KDa and they appeared perfectly fine on the membrane. I really don't know whether there was some problem while I did the blotting with the low molecular weight proteins. I did detect them last time, so it looks like they should work now as well. Thinking of stripping the membranes and reprobing them.
Forgot to add the porosity. Its 0.2um.
you can reversibly stain the membrane for total protein with ponceau s. this and staining the post-transfer gel will allow you to determine if you have efficiently transferred the proteins.