Silver Staining of SDS-PAGE Gels（SDS-聚丙烯酰胺凝胶电泳银染）

1. Rinse gel on glass plate briefly with distilled water.

2. Wash gel in fresh 50% ETOH-12% HAc, with shaking, 3 times, 1 hour each. (Fix IEF gel ON in 10% TCA).

3. Wash gel four times with 10% ETOH-5% HAc for one hour each. One wash may be done overnight.

4. Rinse gel with several changes of water over five minutes.

5. Incubate gel in fresh 200 ml of 0.6 g K2CrO4 (0.0034 M) and 40 µl HNO3 (0.0032 M) for 5 minutes.

6. Rinse gel with several changes of water until color fades, approximately 10 min.

7. Incubate in 150 ml of 0.012 M AgNO3 (0.3 g/150 ml) Illuminated for 30 min, rocking several times to incorporate stain.

8. Rinse gel with three changes of water over 1 minute.

9. Rapidly rinse three times with 100 ml 0.28 M Na2CO3 and 0.5 ml 37% formaldehyde (8.9 g Na2CO3 and 1.5 ml formaldehyde/300 ml). Remove the darkened rinse solution quickly each time.

10. Agitate in 100 ml solution in 9 until desired intensity is reached and then stop development with 100 ml of 3% HAc.

11. Wash twice with water and store in water or in 10%MeOH-3% glycerol. Stain will not fade and can be photographed at your convenience.

12. Wash gel in 50% ETOH and 10% HAc for 2 hours, then in 25% ETOH-10% HAc for 1 hour two times, and then in 10% ETOH-5% HAc for 1 hour twice.

13. Equilibrate gel in 0.012M AgNO3 solution (0.4 g/200 ml) for 2 hours.

14. Rinse with water for 10-20 seconds.

15. Reduce in the following solution for 10 min.: 200 ml 0.75N NaOH (6 g/200 ml) and dissolve 17.5 mg NaBH4 and 1.5 ml 37% Formaldehyde.

16. Wash gel in 0.75% Na2CO3 for 1 hour and store normally. Color will not fade and can be photographed at some time in the future.