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

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
10% resolving	13.3	10	-	16.3	0.4	0.02
12% resolving	16	10	-	13.6	0.4	0.02
15% resolving	20	10	-	9.6	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 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