RE: silver stain
Farzin Gharahdaghi (gharahf@rockvax.rockefeller.edu)
Fri, 4 Jun 1999 08:49:44 -0700
We use silver staining protocol published by A. Shevchenko, M. Wilm, O. Vorm,
and M. Mann, Anal. Chem., 1996, 68, 850-858 routinely without any problems.
We observed that if silver is removed before digestion the MALDI-TOF
analysis enhances the quality of the spectra (F. Gharahdaghi et al
Electrophoresis 20, 601-5, 1999).
There are a few tricks to getting good destaining results.
1. Silver stained gels are usually stored in 1% acetic acid at 4C. The
residual acetic acid should be removed by thoroughly rinsing the gel with
water before destaining.
2. The reducers, potassium ferricyanide (10mg/ml) and sodium
thiosulfate(16mg/ml) should be made fresh. Mix the two reducers in a 1:1
ratio and immediately add the reducers to the gel piece. Once the silver
brown color disappears remove the reducers and wash with water until the
gel piece is clear. (Note: Incubation after washing with water in
ammonium bicarbonate (100mM) at 55C will speed this process.) We make sure
that the gel piece is clear before proceeding with digestion.
Farzin Gharahdaghi
Associate Director
Protein/DNA Technology Center
Rockefeller University
Phone: 1-212-327-7525
FAX: 1-212-327-8620
email: gharahf@rockvax.rockefeller.edu