Negative staining

[P. Friedl]

In article <43tmqh$b3v at mserv1.dl.ac.uk>, Kirsten Blake <KIRST at HMS01.HMS.UQ.OZ.AU> says:
>
>Dear netters,
>Can anyone offer an explanation as to why I get negatively stained 
>bands sometimes and not others when all conditions are the same. 
>
>I am using SDS-PAGE to separate out myosin isoforms. The gel has 6% 
>acrylamide and 30% glycerol in it. The buffer is a standard Lameli 
>buffer. The gels are run at 150V for 2-3 hours at RT. I run two gels 
>in parallel both made from the same gel mixture in the same buffer 
>but in seperate rigs. After silver staining I often find that a 
>sample has negatively stained on one gel but not on the other. Also, 
>samples that stain properly on several occasions every now and 
>then stain negatively. Often every sample on the gel stains 
>negatively but not always.
>
>I am aware of the common things that cause negative staining: Impure 
>water, overloading, metalloproteins,washing away silver reagent; and 
>have ruled out all of them.
>
>Can anyone shed some light onto this for me please!!!
>Thanks in advance.
>
>Kirsten.
>
>       *****************************************
>     *                                           *
>    *   KIRSTEN BLAKE   <kirst at hms01.hms.oz.au>   *
>   *    Dept. of Human Movement Studies            *
>  *     The University of Queensland                *
> *      Qld; 4072                                    *
>  *     AUSTALIA                                    *
>   *                                               *
>    *    Ph: 07 3656822      Fax: 07 3656877      *
>     *                                           *
>       *****************************************
>     
Hello,
negative staining occurs if the redox potential of the 
gel matrix is lower than that of the protein band. If this
occurs, you should use Farmer's reducer for a complete destaining 
of the gel and then perform a second silverstain. Usually there 
is no more negative staining. 
Farmer's reducer 	2%[w/v] potassiumhexacyanoferrat
			3%[w/v] sodiumthiosulfate
			mix prior to use 1:1
bye
P.F.