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SDS-PAGE protein gels
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[[Keating:Experimental Protocols|back to Experimental Protocols]]


recipes
== [[SDS]]-PAGE protein gels ==
 
*written by [[user:ziz|Nora]]
 
'''recipes'''
 
'''2x loading buffer'''


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


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


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


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


To make 5 acrylamide gels:
'''To make 5 acrylamide gels:'''
wash 5 glass plates and 5 white plates with ethanol
 
get 10 side spacers and 5 well spacers
*wash 5 glass plates and 5 white plates with ethanol
stack components in multiple gel caster in order:
*get 10 side spacers and 5 well spacers
top caster ___
*stack components in multiple gel caster in order:
glass plate       |
 
2x side spacer | x5
top caster, 5x (glass plate, 2x side spacer, white plate), glass plate, bottom caster
white plate       |
 
weigh paper __|
*clip caster in place
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
make resolving gel and stacking gel solutions with following recipes
 
{| align="center" border="1"
| 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
*pour the resolving gel into the gel caster
add 200 ul of N-butanol to each gel to smooth out surface
*add 200 ul of N-butanol to each gel to smooth out surface
wait ~20 min to let solidify
*wait ~20 min to let solidify
pour out butanol and rinse thoroughly with water
*pour out butanol and rinse thoroughly with water
add the stacking gel to each gel and insert well spacer
*add the stacking gel to each gel and insert well spacer
let solidify
*let solidify
release clips carefully to prevent bubbles in gel
*release clips carefully to prevent bubbles in gel
wash off excess gel
*wash off excess gel
wrap gels in wet paper towels and plastic wrap
*wrap gels in wet paper towels and plastic wrap
store at 4 C for no more than 2 weeks
*store at 4 C for no more than 2 weeks


To run gels:
'''To run gels:'''
make aliquots of protein samples
*make aliquots of protein samples
dilute with 2x SDS buffer
*dilute with 2x SDS buffer
make enough for loading 15 ul/well for 15 well spacers
*make enough for loading 15 ul/well for 15 well spacers or 20 ul/well for 10 well spacers
          20 ul/well for 10 well spacers
*boil samples at 95 C for 5 min
- boil at 95 C for 5 min
*take out a gel from fridge and remove well spacer
- take out a gel from fridge and remove well spacer
*clean out excess gel from top of gel
- clean out excess gel from top of gel
*clip into gel apparatus and pour in 1x SDS running buffer in reservoirs
- clip into gel apparatus and pour in 1x SDS running buffer in reservoirs
*load samples into wells, including 5 ul of marker
- 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 at 10-15 amps until samples past stacking gel, then run at 20-25 amps
*run until dye at bottom of the gel
- run until dye at bottom of the gel
*remove gel from apparatus and stain
- remove gel from apparatus and stain


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

Latest revision as of 07:49, 8 April 2009

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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, 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
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