Hello everyone,
Thank you for your replies to the question about very high background after
silver staining of Amersham's plastic-backed 2D gels.
Here is a summary of the answers:
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---- There is one reason that I know of that will cause the high background with silver on backed gels. It is silver deposition between the backing sheet and the gel; usually only a problem with diamine silver stains (silver, NaOH and ammonia). If this is the type of stain that you are using, try switching to a regular silver nitrate stain. They are not quite as sensitive, but they work with backed gels. ------- To get rid of background we have used a sodium thiosulphate in very low concentration. First: on a stage of pre-care before silver reagent, after fix reagent. It's 100mg per 500ml. Second: we added it to developer - 2 or 3 crystals per 200ml of developer.------- Using silver staining reactions on plastic backed gels the reagents can only reach the proteins from one side. These reactions are diffusion controlled. Compared to unbacked gels the reaction times must be at least doubled to reach the same equilibrium. The washing steps after the sensitizing step are very important to increase and addition of further washing steps could be necessary to obtain low background.
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Following suggestion 3 from above we have modified our staining protocol. In a nutshell: we now do both the fixation and sensitising step twice. The numbers of all subsequent washing/staining steps are doubled and we also double the time for each step. This procedure takes longer but gives excellent results.
Regards,
Klaus
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