Re. Sensitive Protein Stains
Dr E. Buxbaum
EB15 at le.ac.uk
Mon Mar 24 07:52:36 EST 1997
r.r.d.croy at durham.ac.uk (Dr Ron Croy) wrote:
>We need to use a sensitive protein stain to detect protein spots on 2D
>gels with the intention of sequencing these from PVDF blots (automated
>ABI 477A). Coomassie blue stains are out because they tend to interfere
>with the sequencing and clog up the reaction vessel. Silver staining
>methods are out due to the protein modification.
You can stain the proteins on the blot with Ponceau S solution in acetic
acid. The blotts are differentiated by rinsing them with water, further
rinses remove the stain completely.
You can also try fluorescent staining in the gel, using Nile Red or the
new Sypro stains from Molecular Probes (I haven't tried the latter
because of their high price).
A third method would be negative staining by metal-SDS precipitates.
Incubate the gel with Zn/Imidazole or CuSO4 solutions. This will result
in clear bands against a turbid background, best seen by holding the gel
against a black surface. The metal ions can be removed completely by
incubation in EDTA solution. There has been a publication recently in
Anal. Biochem. on using Methyl trichloroacetate for the same purpose, I
haven't tried this though.
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