Keating:Experimental Protocols:SDS-PAGE
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SDS-PAGE protein gels
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 ___
glass plate |
2x side spacer | x5
white plate __|
glass plate
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
for 5 gels | 30% protogel (37:1) | 1.5M Tris pH 8.8 | 1M Tris pH 6.8 | water | 10% APS | TEMED |
3% stacking | 1.6 | - | 4 | 10.4 | 0.1 | 0.01 |
8% resolving | 10.7 | 10 | - | 10.4 | 0.4 | 0.02 |
8% resolving | 10.7 | 10 | - | 10.4 | 0.4 | 0.02 |
8% resolving | 10.7 | 10 | - | 10.4 | 0.4 | 0.02 |
8% resolving | 10.7 | 10 | - | 10.4 | 0.4 | 0.02 |
8% resolving | 10.7 | 10 | - | 10.4 | 0.4 | 0.02 |
8% resolving 10.7 10 18.9 0.4 0.02 10% resolving 13.3 10 16.3 0.4 0.02 12% resolving 16 10 13.6 0.4 0.02
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