Difference between revisions of "GST-EEA1 Pulldown from Yeast"
From Bridges Lab Protocols
Davebridges (Talk | contribs) (Created page with 'enter protocol here') |
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| + | == '''Materials''' == | ||
| + | *2x HNG (100mL). Combine 100mM HEPES (10mL), 300mM NaCl (7.5mL), 20% glycerol (20mL) | ||
| + | *1x HNG (10mL). Mix 5mL 2xHNG and 1 PI tablet. | ||
| + | *Lysis Buffer (10mL). Combine 5mL 1x HNG, 1 PI tablet, 1mL of 1% NP40, and 50µL of magnesium chloride. | ||
| + | *Glutathione sepharose beads | ||
| + | *Glass beads | ||
| + | |||
| + | ---- | ||
| + | |||
| + | == '''Protocol''' == | ||
| + | *Inoculate cells in appropriate media overnight. | ||
| + | *Re-suspend cells in 1mL of lysis buffer. | ||
| + | *Lyse cells: place approximately 1mL of glass beads into sonicator tube (Weisman Lab) and run in the sonicator 3x for 20 seconds. (Invert tube in between each round) | ||
| + | *Centrifuge at 4°C for 10 minutes. | ||
| + | *Add 1.5mL of 1x HNG to GST and GST-EEA1 proteins. (-80°C) | ||
| + | *Combine 450µL of protein with 450µL of lysates. | ||
| + | *Place tubes end over end for 30 minutes at 4°C. | ||
| + | *Add 50µL of glutathione sepharose beads to each tube. | ||
| + | *Wash each tube with 1x HNG five times at 4°C. | ||
| + | *Load in a 4-12% gel. | ||
Revision as of 18:47, 28 June 2010
Materials
- 2x HNG (100mL). Combine 100mM HEPES (10mL), 300mM NaCl (7.5mL), 20% glycerol (20mL)
- 1x HNG (10mL). Mix 5mL 2xHNG and 1 PI tablet.
- Lysis Buffer (10mL). Combine 5mL 1x HNG, 1 PI tablet, 1mL of 1% NP40, and 50µL of magnesium chloride.
- Glutathione sepharose beads
- Glass beads
Protocol
- Inoculate cells in appropriate media overnight.
- Re-suspend cells in 1mL of lysis buffer.
- Lyse cells: place approximately 1mL of glass beads into sonicator tube (Weisman Lab) and run in the sonicator 3x for 20 seconds. (Invert tube in between each round)
- Centrifuge at 4°C for 10 minutes.
- Add 1.5mL of 1x HNG to GST and GST-EEA1 proteins. (-80°C)
- Combine 450µL of protein with 450µL of lysates.
- Place tubes end over end for 30 minutes at 4°C.
- Add 50µL of glutathione sepharose beads to each tube.
- Wash each tube with 1x HNG five times at 4°C.
- Load in a 4-12% gel.
