Silver Staining Protocol

[Dima Klenchin]

In article <31C676E8.51CB at ucmp.umu.se>,
   Michael Daws <Michael.Daws at ucmp.umu.se> wrote:
->Does anybody have a good protocol for silver staining they could let me 
->have (or reference)?  I have the maniatis protocol, but wonder if there 
->are any recent improvements to the protocol that I should be aware of.  
->I don't want to buy a kit!  Thanks for your help.
->
->Mike Daws

The following is our standard lab procedure. I like it. It's way cheaper
than the same kit from Bio-Rad.


Silver staining protocol
************************

(identical to "Silver Stain Plus" from Bio-Rad and based on Anal. Biochem, 
1987, 165:33-37) 

I. STOCK SOLUTIONS

	  compound			concentration		volume

	- NH4NO3/AgNO3 		       2% each in water		100 ml
	- Tungstosilicic acid 	       10% water		100 ml
	- Formaldehyde 		       3% in water		100 ml
	- Na2CO3 		       5% in water		1 l 

Stocks may be stored at +4oC for at least 1 year. 

II. PROCEDURE

NB: The gel to be stained should NEVER be in contact with hands or metal 
objects. Wear rinsed gloves. 

1. Fixation
	Fix standard minigel in 200 ml 40% methanol, 10% glacial acetic 
acid, 2.5% glycerol for 20 min. with shaking. For larger, thicker, more 
gels increase the volume or time. Rinse the gel in 200 ml water twice for 
10 min. each. 

2. Staining
	Add to 35 ml of water the following in the following order:

	5 ml NH4NO3/AgNO3 stock
	5 ml tungstosilicic acid stock 
	5 ml formaldehyde stock 

Mix well. Pour 50 ml Na2CO3 stock into 100-200 ml beaker with stirring 
bar. While stirring fast, slowly add the above mixture. After all of it is 
added, immediately decant the rinse water from the gel, pour staining 
solution and stain with agitation 10-20 min. or until the desired bands 
intensity is reached. (Note: the same 100 ml will work for 2 or even 3
gels at a time). 

3. Stopping. 
	Decant staining solution and add 100-200 ml 5% acetic acid or 
fixative diluted twice with water. After 5 min. the gel could be put in 
gel drying solution or discarded depending on the result.