During the transfer step from the acrylamide gel to PVDF membrane, my transfer buffer turns dark brown, ringlets of brown circles are present on the actual gels mimicking the mesh on the transfer cassette, and the gel itself turns brown. Despite this, the blots are actually OK (I have been getting some really good results with them) However, I am embarassed and have to keep replacing the transfer buffer, which is costly.
This has happened consistently 4 times now. Each time I changed the buffers and reagants. Is it possible that a contaminant cannot be rinsed out of a electro transfer system? For example, when I use a biorad transfer system for 2 minigels, 250 mA ovenight, no problem. But when I use a Hoeffer transfer tank (with capacity for 16 minigels) this happens every time now (same, 250 mA overnight).
The strange thing about this is that I have not altered my recipes for YEARS and it only happens in the big transfer tank. By the way, I have used that tank model before with the same buffers/solutions and never experienced any problems.
Here are the parameters:
New transfer buffer (pretty standard, 25 mM TRIS, 192 mM Glycine, 20% Methanol, no SDS)
Polyacrylamide gels are cast fresh with the same exact recipes I have used for the last 4+ years. ALL reagants were replaced after the first brown gel anomaly.
The first brown gel anomaly did happen when a summer student was here with some problems following directions and using the equipment. Ever since she has left I have had this problem. I have replaced every single buffer reagant I can think of....! If this is a contaminant in the actual tank, what is it and how do I fix it?