Keating:Experimental Protocols:SDS-PAGE

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(SDS-PAGE protein gels)
(SDS-PAGE protein gels)
Line 50: Line 50:
*stack components in multiple gel caster in order:
*stack components in multiple gel caster in order:
-
top caster   ___
+
top caster, 5x (glass plate, 2x side spacer, white plate), glass plate, bottom caster
-
glass plate    |
+
*clip caster in place
-
 
+
-
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
*make resolving gel and stacking gel solutions with following recipes
Line 75: Line 65:
| 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
+
| 10% resolving || 13.3 || 10 || - || 16.3 || 0.4 || 0.02
|-
|-
-
| 8% resolving || 10.7 || 10 || - || 10.4 || 0.4 || 0.02
+
| 12% resolving || 16 || 10 || - || 13.6 || 0.4 || 0.02
|-
|-
-
| 8% resolving || 10.7 || 10 || - || 10.4 || 0.4 || 0.02
+
| 15% resolving || 20 || 10 || - || 9.6 || 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
 
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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
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*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:'''
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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
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heat in microwave 30 sec, rocker 10 min, x 2-3
+
*rinse gel with water
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rinse gel with water
+
*add destain and incubate 3-4hr or overnight
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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
+

Revision as of 12:53, 16 September 2005

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
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