| http://axon.med.harvard.edu/~cepko/protocol/mike/G1.html
Solutions
Elution Buffer
50 mM Tris 7.5 5 ml 1M Tris pH 7.5
0.1% SDS 0.1 g SDS
0.1 mg/ml BSA 1.0 mg BSA
0.25 mM EDTA 50 ml 0.5M EDTA
2.5% glycerol 2.5 ml glycerol
up to 100 ml with Q
For every 10 ml add:
10 ml 0.5 M DTT
50 ml 100 mM PMSF (Sigma #P7626)
10 ml Pepstatin A (2.0 mg/ml)
10 ml Leupeptin (1.0 mg/ml)
10 ml Aprotinin (5.0 mg/ml)
(Protease Inhibitor Cocktail Sigma #P8340)
Denaturation Buffer
6 M guanidine hydrochloride 57.3 g G-HCl
20 mM Hepes 7.9 2 ml 1M Hepes pH 7.9
10 mM KCl 0.074 g KCl
0.5 mM DTT 100 ml 0.5 M DTT
up to 100 ml with Q
Dialysis Buffer
20 mM Hepes 7.9 20 ml 1M Hepes 7.9
10 mM KCl 0.74 g KCl
10% glycerol 100 ml glycerol
0.5 mM DTT 1 ml 0.5 M DTT
1 mM PMSF 10 ml 100 mM PMSF
up to 1 liter with Q
add DTT and PMSF just before use
Procedure
• Sigmacote several dozen lock top eppendorf tubes. In the hood add approximately 0.5 ml Sigmacote to an eppendorf tube, invert several times and then pour the Sigmacote into the next tube etc... Remove the last traces of Sigmacote for each tube, rinse with Q, and autoclave.
• Run a standard SDS-PAGE gel and cut out the band(s) of interest. Crush the band in eppendorf tube using a blue tip pestle and add 1 ml of ice cold Elution Buffer.
• Tilt overnight at 4°. Spin down gel fragments for10 minutes at 4° and remove the elution buffer. Filter the eluate into another eppendorf tube to give a total yield of approximately 600 ml.
• Put 300 ml into each of 2 Sigmacote treated eppendorf tubes and add 1.2 ml ice cold acetone to each. Spin for 30 minutes at 4°. Wash the pellet in ice cold methanol.
• Resuspend each pellet in 50 ml Denaturation Buffer and dialyze against 330 ml Dialysis Buffer with 2 changes.
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