thanks a lot guys... I had put up this question regarding the poor and uneven transfer that I was getting from my gel onto my PVDF membrane last week. I was having problem with proteins being left on the gel after transfer. Well.. I solved that problem after getting tips and pointers from the members of this newsgroup. Thanks a lot. But I encountered another problem too when I solved this one (that is life I guess!!).. I need your suggestions.
Even though SDS does sort of inhibit binding of proteins to PVDF and methanol improves it to some extent, I thought the best way to overcome uneven transfer across the blot was to achieve a complete transfer of proteins from the gel (as some members suggested too) and hence overcome the problem of uneven transfer rates across the blot. So I used a Tris-glycine buffer with 0.05% SDS and 10 % methanol and a control was also maintained with the standard buffer we use in our lab (Tris glycine buffer with 20 % methanol, NO SDS). I use a mini transblot aparatus (tank system) and a 8 x 5 cm gel (15 % SDS PAGE). Results were as follows.
- High amount of flow through (nearly 40 %) in the system using SDS and 10 % methanol but VERY UNIFORM (thats what I need) transfer of bands across the length of the blot (equal amounts of proteins had been loaded in each well).
- No flow through in the control blot using 20 % methanol and no SDS, but very uneven transfer across the blot.
Well,, the high flow through is a problem, but the even transfer is good. Even though I know that I cant eliminate the flow through as long as there is SDS in the transfer buffer, What would your suggestions be to achieve a better binding of proteins onto blot and at the same time achieve a maximal and even transfer of proteins onto blot???
I would value your suggestions. thanks everybody again.. YOu guys are swell.