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nucat 09-10-2010 09:45 PM

Acrylamide gel turns brown during transfer...?
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?

Thank you!

MJD 09-13-2010 08:59 PM

Re: Acrylamide gel turns brown during transfer...?
Strange phenomena. You could always rinse the Hoefer tank with 10% bleach to kill any organisms growing inside it. It is definitely not your reagents, since it doesn't happen in the Biorad tank. Good Luck!

luisillo 09-14-2010 07:56 PM

Re: Acrylamide gel turns brown during transfer...?
That reminds me of the silver staining protocol of polyacrilamide gels I used to use. The developing solution turns dark when the silver sol. (AgNO3 diluted in 10% EtOH + 0.5% acetic acid) makes contact with it. The developing solution turns the gel brown and its composed of 3% NaOH and 0.5% formaldehide.

What this has to do with your problem, well, there might be something in your tank that is making a chemical reaction to occur to the gel, probably a metal.

blazepi13 02-26-2011 12:36 AM

Re: Acrylamide gel turns brown during transfer...?
Are you using Tris HCl instead of Tris base?

nucat 02-26-2011 06:44 AM

Re: Acrylamide gel turns brown during transfer...?
No...and strangely enough, when I use biorad tank (2 blots )this does not happen, only with Hoeffer large tank (can hold up to 6 blots) does it occur! I called Hoeffer and they also seem mystified! It still happens, but I have lovely Western blots, so I guess other than being unsightly and constantly making new buffer, it has no real consequence to the experiments. Would still like to know why it happens though.

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