PAGE - Silver Stain

[Paul N Hengen]

I am doing SDS-PAGE using the Tris Tricine buffer system of Schagger with
a 10% polyacrylamide/4% stacker gel.

@article{Schagger1987,
author = "H. Schagger
     and G. von Jagow",
title = "Tricine-Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis
for the Separation of Proteins in the Range from 1 to 100 kDa",
journal = "Anal. Biochem.",
volume = "166",
pages = "368-379"
year = "1987"}

Following the run, I fixed in 50% MeOH/10% acetic acid O/N, then in 10% MeOH/7%
acetic acid for 30 min., and then in MilliQ water for approx. 2 hrs, before
staining with the Silver Stain Plus kit from Bio-Rad. The first time I did
this, I got a very sensitive signal, but there was a little bit of background
as well as some grainy spots on the gel. Then I read that soaking for 30 min.
in 10% unbuffered glutaraldehyde could reduce the background according to the
authors of this paper:

@article{Oakley1980,
author = "B. R. Oakley
     and D. R. Kirsch
     and N. R. Morris",
title = "A Simplified Ultrasensitive Silver Stain for Detecting
Proteins in Polyacrylamide Gels",
journal = "Anal. Biochem.",
volume = "105",
pages = "361-363",
year = "1980"}

When I tried this, I got absolutely no silver stained proteins (no bands).

Questions:

1. What does glutaraldehyde do to reduce the background in silver stained gels?

2. Why did I get a completely clean gel after silver staining?

Any other suggestions are welcome.

*******************************************************************************
* Paul N. Hengen, Ph.D.                           /--------------------------/*
* National Cancer Institute                       |Internet: pnh at ncifcrf.gov |*
* Laboratory of Mathematical Biology              |   Phone: (301) 846-5581  |*
* Frederick Cancer Research and Development Center|     FAX: (301) 846-5598  |*
* Frederick, Maryland 21702-1201 USA              /--------------------------/*
*******************************************************************************