Silver Staining of Proteins Formaldehyde Fixation

Colors and intensity of bands may differ after formaldehyde fixation compared with those obtained by glutaraldehyde fixation. Formaldehyde yields blackish and glutaraldehyde gives brownish bands. 

4 ml of 25% ammonia and 4.0 ml 1 N NaOH are filled up to 16.0 ml with ddH20, then 4.0 ml 10% AgNOs (w/v) are added dropwise with shaking. The precipitate formed by a drop has to dissolve completely before giving the next. If the last drops are not dissolved completely, some ammonia can be added carefully. The resulting solution is pale brown. When the addition of silver is complete, ddH20 is added to 100 ml. 

The solution is prepared freshly before using. After use, the solution has to be destroyed by hydrochloric acid. 

The gel is fixed for 1 h in Soln. A. Wash the gel at least four times with water about 30 min for each wash and preferably overnight for the last wash. Agitate slowly with each wash. Stain the gel with at least five gel volumes Soln B (to cover the gel) for 30 min with shaking. Pour off the silver solution, wash with ddH20 four times for 5 min each and develop with Soln. C as described in Protocol 2.4.2.I. Development is stopped after decanting Soln. C by Soln. D. The gel is stored in Soln. D until drying. 

Ochs DV, McConkey EH, Sammons DW (1981) Electrophoresis 2 304 Yiiksel KU, Gracey RW (1985) Electrophoresis 6 361 

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