Pierce Silver Stain Kit
Wash gel in ultra pure water for 5 minutes.
Replace the water and wash for another 5 minutes.
Fix gel in 30% ethanol:10% acetic acid solution (i.e., 6:3:1 water:ethanol:acetic acid) for 15 minutes.
Replace the solution and fix for another 15 minutes.
Note: Gel may be kept in fixing solution overnight without affecting stain performance.
Wash gel in 10% ethanol solution for 5 minutes.
Replace solution and wash for another 5 minutes.
Wash gel in ultrapure water for 5 minutes.
Replace water and wash for another 5 minutes.
Prepare Sensitizer Working Solution by mixing 1 part Silver Stain Sensitizer with 500 parts ultrapure water (e.g., mix 50µL Sensitizer with 25mL water).
Incubate gel in Sensitizer Working Solution for exactly 1 minute, then wash with two changes of ultrapure water for 1 minute each.
Prepare Stain Working Solution by mixing 1 part Silver Stain Enhancer with 50 parts Silver Stain (e.g., 0.5mL of Enhancer with 25mL Stain).
Incubate gel in Stain Working Solution for 30 minutes.
Note: Gel may be incubated in Stain Working Solution for as short as 5 minutes or as long as overnight without affecting stain performance.
Prepare Developer Working Solution by mixing 1 part Silver Stain Enhancer with 50 parts Silver Stain Developer (e.g., mix 0.5mL of Enhancer with 25mL Developer).
Prepare 5% acetic acid solution as a Stop Solution.
Quickly wash gel with two changes of ultrapure water for 20 seconds each.
Immediately add Developer Working Solution and incubate until protein bands appear (2-3 minutes).
Note: Protein bands will begin to appear within 30 seconds and then continue to develop.
Between 2 and 3 minutes, protein detection vs. background is optimal.
After 3 minutes, lane background signal may increase to undesirable levels.
When the desired band intensity is reached, replace Developer Working Solution with prepared Stop Solution (5% acetic acid).
Wash gel briefly, then replace Stop Solution and incubate for 10 minutes.
