(1)         All containers must be cleaned (preferably with nitric acid) and rinsed well with distilled/Millipore water.

(2)         All solutions must be prepared in distilled/Millipore water.  Try to prepare all solutions fresh and have them ready to go.

(3)         After separation of proteins by SDS-PAGE, place the gel in a glass vessel.  All incubation should be done with constant shaking (preferably a belly dancer).

(4)         Fix the gel/proteins in 250 ml of 50% methanol (30 min for gels 1-1.5 mm thick; 15 min for thinner gels).

(5)         Rinse the gel in 250 ml of 5% methanol (30 min for gels 1-1.5 mm thick; 15 min for thinner gels).

(6)         Reduce proteins by incubating the gel in 250 ml of 0.1 mM DTT in distilled/Millipore water (30 min for gels 1-1.5 mm thick; 15 min for thinner gels).

(7)         Incubate the gel in 250 ml of 0.2% AgNO3 (to 245 ml distilled water, add 5 ml of 10% AgNO3 stock purchased from VWR).  Incubate gel in silver solution 30 min for gels 1-1.5 mm thick; 15 min for thinner gels.

(8)         Wash briefly 3 times with distilled/Millipore water (3 x 10 seconds).

(9)         Wash briefly with 150 ml developing solution (see below).

(10)     Develop gel/bands in 350 mM Na2CO3/0.02% formaldehyde (to make 500 ml developing solution: dissolve ~15 g Na2CO3 in distilled/Millipore water, then add 0.25 ml 37% formaldehyde).

(11)     Develop until bands are of desirable intensities (usually take a 1-5 minutes).  Overdeveloping will increase background.

(12)     Stop by transferring the gel to a new glass vessel containing 250 ml of 5% acetic acid (use cleaned and rinsed gloves when transferring gel).

(13)     Gel can be stored in 5% acetic acid or rinse with water before drying.