Hello,
I have consistently had issues with uneven western blot transfers at the lower MW regions (as seen in the attached picture). The transfer system is the Thermo Mini Blot Module Wet transfer system. Transfer buffer contains Tris, Glycine, and 20% methanol. Transfer conditions were 25V for 1 hour. This phenomenon was seen with multiple gel percentages. Any insights on how to fix this issue?
*** Same sample was loaded equally in all lanes
Thank you
For WB 10% ethanol in western blot transfer buffer works well and 100% ethanol for wetting the membrane.
Also dry or seminary transfer work well reducing waste. Or alcohol free transfer Dunn (doi:10.1016/0003-2697(86)90207-1, 10 mM NaHCO3, 3 mM Na2CO3, with 50 µM SDS for large transmembrane proteins). Alcohol is more important for small proteins.
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