Difference between revisions of "Amylose pull-down of glycogen-bound proteins"
From Bridges Lab Protocols
(Created page with '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 i...') |
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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
