Silver staining problem

Tom Thatcher ttha at
Sat Feb 10 22:45:25 EST 1996

In <4fj93s$80q at> hamburg at (Eric Sarnighausen) writes:

>I am analyzing patterns of cell wall proteins by two dimensional
>electrophoresis. Unfortunately, the most prominent protein of 23 kDa
>(that I am extremely interested in) is not silver stained at all (using
>the acidic method). 

I have not had that specific problem but here is my favorite silver stain.
Sometimes a different technique gives different results, and everyone I've
shared this with goes bonkers over it.

Silver Staining of SDS Protein Gels

Reference: Lischwe and Ochs, 1982. Anal. Biochem. 127:453-457.

All operations should be carried out in glass trays. If the gel is 
touched, fingerprints will appear when it is developed.

1.	Fix the gel in 50% ethanol, 10% acetic acid at least 1 hour, with 
gentle shaking.

2.	Wash the gel in 10% ethanol, 5% acetic acid, 2 times for 30 minutes 
each, with gentle shaking.

3.	Incubate the gel in 100 ml 0.02% AgNO3 (0.2 g AgNO3 per 100 ml H2O) 
for 1 to 2 hours, with gentle shaking

4.	Aspirate or pour the silver solution into an appropriate waste 
container. Rinse the gel for a few seconds in distilled H2O.

5.	The developer is 4.5 g NaOH, 13 mg NaBH3 (sodium borohydride), and 1 
ml formaldehyde (commercial stock solution, 37% formaldehyde) in 150 
ml H2O. Develop the gel. Pour a few ml developer into the tray with 
the gel. A black precipitate will form. Quickly aspirate away this 
precipitate, then add the rest of the developer. Optimum developing 
time for a 0.75 mm thick gel is 4 to 5 minutes; slightly longer for 
thicker gels.

6.	Incubate the gel in 100 ml 0.75% Na2CO3 for three changes of 30 
minutes each. This enhances and stabilizes the silver stain. The 
complete treatment is not absolutely necessary, but the gel should be 
treated at least once.

7.	Soak the gel in 3% acetic acid to stop further development. The gel 
will eventually fade (in a few days) unless it is dried down.

Tom Thatcher                          | You can give a PC to a Homo habilis,
University of Rochester Cancer Center | and he'll use it, but he'll use it
ttha at           | to crack nuts.

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