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. |
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