Hello,
I have issues with transferring 1.5mm thick SDS-PAGE gels (14% acrylamide, with saccharose) on PVDF membrane. I tried 50 min @ 100V in Towbin buffer with 10% Met-OH, but most of the proteins stayed on the gel (I stained both gel and membrane).
I read about ON transfer @ 30V/90mA, but I am not sure which on e should be constant. Did you do this transfer in your lab?
What would you recommend?
Hello Malgorzata, just to indicate that I do transfers routinely from 1.5 mm acrylamide gels (without saccharose) and they work perfectly. However for a transfer at 100V I go for 80 mins but perfect transfers occur O/N at 30V. You may consider trying this. We work with Tris-Gly buffer containing MeOH.
I agree with Robert Adamu Shey , time of transfer depends of protein's size ( big protein's size needs a long time, ususally 60 min 100 V is enough, i use trizma-glycine-etOH transfer buffer for it).Also check pore size of PVDF membrane
Thank you for your replies! I tried 80 min @ 100V and it worked great. Seems transfer duration was the case. Thanks again!
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