Keating:Experimental Protocols:SDS-PAGE: Difference between revisions
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*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 | ||
*clip caster in place | |||
*clip caster in place | |||
*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 | ||
|- | |- | ||
| | | 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 | ||
*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: | '''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 | |||
Revision as of 10:53, 16 September 2005
| Information concerning the Keating Lab | |
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Resources: | |
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
