We had several times the problem after the transfer: you can detect the yellow 10 kDa Band, the blue 20 kDa Band, the pink 25 kDa Band but after only the pink 75 kDa Band.
15, 37 and 50 kDa are missing??? The marker arrived last week was directly aliquoted and stored in -20°C. Transfer Buffer was 0.025% SDS and 20% Methanol, SDS PAGE Gels were 7.5-12%. Transfer was according to criterion wire Blot guide 100 V, 1h. We were suggesting that the proteins ran through the membrane, but how is it possible that we see the lower Marker Bands? There are no proteins left in the Gel (coomassie) under the 100 kDa Marker Band (which is fine because we are interested in the smaller molecules). So where have the middle markers gone?
Are these bands missing only on the blot, or already in the gel? Is it a problem of visibility (can be solved by, say, doubling the amount of marker per well)? Are you using nitrocellulose or PVDF membranes (the latter shows better protein binding)? Are you using tank or semi-dry blotters? In the latter, heat is a bigger problem than in the former. For optimal transfer, use Dunn's buffer (doi:10.1016/0003-2697(86)90207-1) rather than Towbin's. 100 V sounds a little much, I use 40 V.
Hi Engelbert, in the Gel the marker Looks beautiful! Only on the pvdf membrane after wet Blot Transfer some Bands are missing!
OK, that is half the rent. You only have to optimise the transfer, and I have given you some hints on how to do that. Of course, you should also think of trivial mistakes like air bubbles between gel and membrane. Perhaps the first step would be to stain the blot with Ponceau S (doi:10.1016/0003-2697(86)90263-0) and check whether only the standards are affected or all proteins in that size range.
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