(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
(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%
(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.