protein flowthough in PVDF western

hi..
This is a followup question from a previous problem I had (which I solved thanks to member suggestions). I am getting complete transfer of protein from gel to blot when I do my westerns now. I use Tris-glycine buffer +20 % methanol +0.05% SDS as transfer under ice pak cooling using a mini-transblot aparatus from BioRad (uses small 8 x 5 cm gels) and 90 V for 45 minutes on a magnetic stirrer. I use two PVDF membranes on top of the gel, the primary which I use for antibody decoration and a secondary for checking flowthrough. The primary membranes that I use show good transfer towards the SIDES while slightly less transfer as we go towards the center of the blot. This is very consistent in all blots I run. But the secondary membrane show that the flowthrough proteins (after staining membrane) have increased staining intensity towards CENTER of blot than towards the side. This shows that there is an increased flowthrough of proteins towards the center of the blots and hence lower protein retention towards the center of primary blot.
I need very uniform and even transfer and even protein retention on my blots for my purpose. I believe that I am seeing some sort of temperature gradient across the blot cooler towards the side and warmer towards the center of the blot while transfer. But I am not sure whether temperature affects protein retention on PVDF membrane to that much extent. Am I wrong? Has anyone else seen this effect?
Would reducing the voltage or increasing the time of transfer be useful in solving this problem?
I would appreciate your suggestions in this regard.
thank you
Jai

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I neer used SDS in the transfer buffer, as well as I never had problems with
uneven transfer. Also, if transfering at high voltage (30-100V), I do it in
the cold room, never trusting the ice pack/mixing. Have you tried non-SDS
buffer with 15% MeOH with pre-soaking of the gel in that buffer before
transfer as I suggested before? If you insist on room temperature transfers,
try doing it at max. 15 V overnight.

If you want to fing out whether your uneven transfer is due to SDS presence
in the buffer, increase SDS conc. to say 1% and do the trensfer in parallel
with the same gel control being transfered in 0.5% and 0% SDS buffer.
Emir

""Jayakumar, R"" <[Only registered users see links. ]> wrote in message
news:97101976F8A044468CA74FE11883B90E02048922@VIST A.roswellpark.org...
hi..
This is a followup question from a previous problem I had (which I
solved thanks to member suggestions). I am getting complete transfer of
protein from gel to blot when I do my westerns now. I use Tris-glycine
buffer +20 % methanol +0.05% SDS as transfer under ice pak cooling using a
mini-transblot aparatus from BioRad (uses small 8 x 5 cm gels) and 90 V for
45 minutes on a magnetic stirrer. I use two PVDF membranes on top of the
gel, the primary which I use for antibody decoration and a secondary for
checking flowthrough. The primary membranes that I use show good transfer
towards the SIDES while slightly less transfer as we go towards the center
of the blot. This is very consistent in all blots I run. But the secondary
membrane show that the flowthrough proteins (after staining membrane) have
increased staining intensity towards CENTER of blot than towards the side.
This shows that there is an increased flowthrough of proteins towards the
center of the blots and hence lower protein retention towards the center of
primary blot.
I need very uniform and even transfer and even protein retention on my
blots for my purpose. I believe that I am seeing some sort of temperature
gradient across the blot cooler towards the side and warmer towards the
center of the blot while transfer. But I am not sure whether temperature
affects protein retention on PVDF membrane to that much extent. Am I wrong?
Has anyone else seen this effect?
Would reducing the voltage or increasing the time of transfer be
useful in solving this problem?
I would appreciate your suggestions in this regard.
thank you
Jai

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