Problem with silver staining

sudheer sangeetham via methods%40net.bio.net (by sudheer.pbm07 from gmail.com)
Wed Feb 22 12:13:35 EST 2012


Hi All
I am doing silver staining of my protein,which exposed to UV light, the
method I am giving here

1. Fixing Solution ( Methanol - 50%, Glacial Acetic Acid-12%,Formaldehyde
(37%) - 0.0185% )........ > 1 hr.
2. Wash 50% ethanol 2 x 20 min.
3. Wash 30% ethanol 20 min.
4. Pretreatment Solution (Na2S2O3. 5H2O- 0.2mg/ml)  exactly 1 min.
5. Rinse with diH2O exactly 3 x 20 sec.
6. Impregnate Silver Solution (AgNO3 - 2mg/ml, Formaldehyde (37%) - 0.0278%
- 20 min.
7. Rinse diH2O exactly 2 x 20 sec.
8. Developing Solution ( Na2CO3 - 60 mg/ml , Na2S2O3.5H2O - 4µg/ml,
Formaldehyde (37%) -  0.0185%...till you could see the bands

9. Stop Solution (Methanol - 50%, Glacial Acetic Acid-12% ).

In this experiment my aim is to see higher molecular weight band of
protein, which exposed to UV, on top of the gel. In my case negative
control sits at original place and nothing could be seen at higher mol
weight but incase of sample exposed to UV, some of the protein disappeared
at original position but I couldnt see anything on the top of gel.

If any one give any suggestions that would be greater help.

Thank you in advance

-- 
Sudheer Babu.S
Ph.D student
Institute of Biochemistry
Biological Research Center
Szeged,Hungary.


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