High background with SDS

Jens Krieger krieger at mibm1.ruf.uni-freiburg.de
Thu Jul 4 16:57:18 EST 1996


[ Article crossposted from bionet.molbio.methds-reagnts ]
[ Author was Jens Krieger (krieger at mibm1.ruf.uni-freiburg.de) ]
[ Posted on 4 Jul 1996 21:55:29 GMT ]

Hello!
I'm staining SDS-PAGE with methylgreen, using the method of Cutting and 
Roth (73), but I've problems with high background and no seriously fixed 
proteins.
I fix the gels with sulfosalicylic acid (SSA, 10% w/v)) and CaCl2 to build 
unsoluble calcium-phosphat complexes. So far, so good. With the last 
step, rinsing with aequos ammonium-molybdat, there should be 
phospho-molybdat complexes. I think it works so far, because if I stain 
it with methylgreen (5% in 7% acetic acid) and then destain it with SSA, 
i will see for a short time very sharp green bands, but there are still a 
very high background. So I have to destain ti further on, and 
suddenly....the bands disappear. The background is okay, but without any 
bands.....
Does everyone has an idea, why the bands suddenly disappear? Another 
destaining reagent, another fixing reagent? (i've tried TCA and normal 
acetic acid; it doesnt work at any time).

Thanks in advance, Jens.

--
/~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~\
|  Jens Krieger		"Solange die Alpen noch stehen, |
|  				wird es keine		|
|  Tel.:+49.351.463.5465  	   Gerechtigkeit geben."|
|							|			
|  krieger at rcs.urz.tu-dresden.de	H.Achternbusch	|
\  krieger at mibm.ruf.uni-freiburg.de			/
 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

--
/~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~\
|  Jens Krieger		"Solange die Alpen noch stehen, |
|  				wird es keine		|
|  Tel.:+49.351.463.5465  	   Gerechtigkeit geben."|
|							|			
|  krieger at rcs.urz.tu-dresden.de	H.Achternbusch	|
\  krieger at mibm.ruf.uni-freiburg.de			/
 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~



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