Keating:Experimental Protocols:SDS-PAGE

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SDS-PAGE protein gels


2x loading buffer 100 mM Tris-Cl pH 6.8 4% SDS 0.2% bromophenol blue 20% glycerol 200 mM DTT (add right before using)

5x Tris-glycine running buffer 25 mM Tris 250 mM glycine pH 8.3 0.1% SDS

Coomassie stain (1L) 2.5 g Coomassie dye 500 ml methanol 400 ml water 100 ml glacial acetic acid

destain (1L) 500 ml methanol 400 ml water 100 ml glacial acetic acid

To make 5 acrylamide gels: wash 5 glass plates and 5 white plates with ethanol get 10 side spacers and 5 well spacers stack components in multiple gel caster in order: top caster ___ glass plate | 2x side spacer | x5 white plate | weigh paper __| bottom caster clip caster in place – don’t add extra to make tighter, better to let it leak make resolving gel and stacking gel solutions with following recipes

pour the resolving gel into the gel caster add 200 ul of N-butanol to each gel to smooth out surface wait ~20 min to let solidify pour out butanol and rinse thoroughly with water add the stacking gel to each gel and insert well spacer let solidify release clips carefully to prevent bubbles in gel wash off excess gel wrap gels in wet paper towels and plastic wrap store at 4 C for no more than 2 weeks

To run gels: make aliquots of protein samples dilute with 2x SDS buffer make enough for loading 15 ul/well for 15 well spacers

          20 ul/well for 10 well spacers

- boil at 95 C for 5 min - take out a gel from fridge and remove well spacer - clean out excess gel from top of gel - clip into gel apparatus and pour in 1x SDS running buffer in reservoirs - load samples into wells, including 5 ul of marker - run at 10-15 amps until samples past stacking gel, then run at 20-25 amps - run until dye at bottom of the gel - remove gel from apparatus and stain

To stain gels with Coomassie: add gel and some Coomassie stain to an empty pipette tip box incubate on rocker overnight or heat in microwave 30 sec, rocker 10 min, x 2-3 rinse gel with water add destain and incubate 3-4hr remove destain and add water to let gel enlarge take picture in Bell lab w/ transluminating white light