Haystead Lab Silver Stain Protocol

1. Fix gel in 10% MeOH, 10% acetic acid for 30 minutes or O/N.
2. Wash gel 4X in water, at least 5 min. per wash. Gels cannot be overwashed.
3. Incubate gels in sodium thiosulfate (1 or 2 pellets (~400mg) per 500 ml of water) for 90 seconds exactly! Save 20 ml of this sodium thiosulfate solution for the developing solution.
4. Wash gels 3X quickly with water. (Fill up dish with gels with water, and then dump out the water at the sink.)
5. Add silver nitrate solution (0.9 g silver nitrate in 500 ml water) and let gels stain for 10 min. Gels should turn slightly yellow. Silver nitrate can be recovered and reused.
6. Wash gels 3X quickly with water. (Fill up dish with gels with water, and then dump out the water at the sink.)
7. Add developer solution (10 g potassium carbonate, 20 ml sodium thiosulfate solution from step 3, and 250 µL 40% formaldehyde in 500 ml).
8. Stop reaction by addition of destain (10% MeOH, 5% acetic acid).
9. Wash gels in water, 2-3X, and leave in water.

Click here for guidance on selecting bands to be sequenced

References:

Graves, PR and Haystead T.A.J. (2002) Molecular Biologists Guide to Proteomics. MMBR, 66(1):39-63.

Mackey AJ, Haystead TA, Pearson WR.Getting more from less: algorithms for rapid protein identification with multiple short Peptide sequences. Mol Cell Proteomics. 2002 Feb;1(2):139-47.

MacDonald JA, Mackey AJ, Pearson WR, Haystead TA. A strategy for the rapid identification of phosphorylation sites in the phosphoproteome. Mol Cell Proteomics. 2002 Apr;1(4):314-22.

Graves P, Kwiek J, Fadden P, Ray R, Hardeman K, Coley A, Foley M and Haystead TAJ (2002). Discovery of novel antimalarial targets in the human purine binding proteome. Mol Pharm 62(6):1364-1372.

MacDonald JA, Borman MA, Muranyi A, Somlyo AV, Hartshorne DJ, Haystead TA. Identification of the endogenous smooth muscle myosin phosphatase-associated kinase. Proc Natl Acad Sci U S A. 2001 Feb 27;98(5):2419-24.

Alms GR, Sanz P, Carlson M and Haystead TAJ. (1999) Reg1p targets protein phosphatase 1 to dephosphorylate hexokinase II in Saccharomyces cerevisiae: Characterizing the effects of a phosphatase subunit on the yeast proteome. EMBO J.18 (15):4157-4168.

Damer CK, Partridge J, Pearson WR, Haystead TA. Rapid identification of protein phosphatase 1-binding proteins by mixed peptide sequencing and data base searching. Characterization of a novel holoenzymic form of protein phosphatase 1.J Biol Chem. 1998 Sep 18;273(38):24396-405.