Amylose pull-down of glycogen-bound proteins: Difference between revisions
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All steps on ice/cold room | ==Protocol== | ||
Add 50 ul beads (amylose resin #E80021L Biolabs) to 0.5 mg of protein lysate in lysis buffer with protease inhibitors | * All steps on ice/cold room | ||
Add 1 ml of lysis buffer | * Add 50 ul beads (amylose resin #E80021L Biolabs) to 0.5 mg of protein lysate in lysis buffer with protease inhibitors | ||
Rotate in cold room 30’ to ON | * Add 1 ml of lysis buffer | ||
Spin down at max speed for 1’ | * Rotate in cold room 30’ to ON | ||
Aspirate supernatant down to 1 mm over beads with crushed tip. | * Spin down at max speed for 1’ | ||
Repeat 3-5 washes with lysis buffer without protease inhibitors | * Aspirate supernatant down to 1 mm over beads with crushed tip. | ||
After last wash and aspiration spin again and aspirate all supernatant | * Repeat 3-5 washes with lysis buffer without protease inhibitors | ||
Ad 50ul SDS loading buffer, boil for 5 minutes | * After last wash and aspiration spin again and aspirate all supernatant | ||
Load 10ul/gel | * Ad 50ul SDS loading buffer, boil for 5 minutes | ||
* Load 10ul/gel | |||
Latest revision as of 13:59, 28 May 2012
Protocol
- All steps on ice/cold room
- Add 50 ul beads (amylose resin #E80021L Biolabs) to 0.5 mg of protein lysate in lysis buffer with protease inhibitors
- Add 1 ml of lysis buffer
- Rotate in cold room 30’ to ON
- Spin down at max speed for 1’
- Aspirate supernatant down to 1 mm over beads with crushed tip.
- Repeat 3-5 washes with lysis buffer without protease inhibitors
- After last wash and aspiration spin again and aspirate all supernatant
- Ad 50ul SDS loading buffer, boil for 5 minutes
- Load 10ul/gel