Keating:Experimental Protocols:SDS-PAGE

From OpenWetWare

Revision as of 15:30, 27 December 2005 by Ziz (Talk | contribs)
Jump to: navigation, search
Information concerning the
Keating Lab
Research

Lab Members

Resources:
Publications
Experimental Protocols
Coder's Corner
Internal

back to Experimental Protocols

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, 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
Personal tools