Silver Staining of SDS-PAGE Gels(Ⅰ)

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.

Color Silver Stain

1.Fix gel in 50% ETOH and 10% HAc for 2 hours or more.

2.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.

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

4.Rinse with water for 10-20 seconds.

5.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.

6.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.