Keating:Experimental Protocols:SDS-PAGE

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SDS-PAGE protein gels

 * written by Nora

recipes

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, 5x (glass plate, 2x side spacer, white plate), glass plate, bottom caster


 * clip caster in place


 * 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 or 20 ul/well for 10 well spacers
 * boil samples 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 or overnight
 * remove destain and add water to let gel enlarge
 * take picture in Bell lab w/ transluminating white light