Silver staining.  This silver staining procedure is more complicated than some others but I find it to be very sensitive.  The proteins stain very darkly.  Also, it works well with gels that have been Formaldehyde-fixed, which is how I prefer to prepare gels that analyze products of protein-DNA crosslinking reactions.

For Formaldehyde-fixed gel:

  1. Immerse gel for 1 hr in 51.4ml EtOH/120ml H2O/28.5ml 35% Formaldehyde/228mg Coomassie.

  2. Soak gel for 1 hr in 62.5ml EtOH/187.5ml H2O/2.5ml 35% Formaldehyde/300mg Coomassie.

  3. Destain gel with 250ml EtOH/750ml H2O/10ml Formaldehyde.

  4. Wash gel with several changes of H2O.

For MeOH/Acetic acid-fixed gel:

  1. Fix gel 30'-60' in 50% MeOH/ 10% HOAC (0.1% coomasie is optional)

  2. Wash in 5% MeOH/ 7% HOAc for >5hr

  3. Rinse gel once in water

  4. Soak gel in 2.5% glutaraldehyde for 30'

  5. Wash gel extensively in water for >3hr

Silver staining:

  1. Prepare fresh silver stain solution:

  2. Soak gel in silver stain solution for 15min.

  3. Wash gel 3 X 5 min. with H2O.

  4. Prepare developer by combining 25mg citric acid/500ml H2O/0.25ml 37% formaldehyde (which contains 10% MeOH).

  5. Soak gel in developer for 10-15' replacing developer when it yellows; addition of MeOH to 5-10% will slow development.

  6. Stop development by washing away developer and adding 50% MeOH.

  7. Wash in water and store sealed in pouch.