Difference between revisions of "GST-EEA1 Pulldown from Yeast"
From Bridges Lab Protocols
(added link to 2xHNG) |
|||
| Line 1: | Line 1: | ||
| − | |||
== '''Materials''' == | == '''Materials''' == | ||
| − | *2x HNG (100mL). Combine 100mM HEPES (10mL), 300mM NaCl (7.5mL), 20% glycerol (20mL) | + | *[[ 2xHNG_Buffer | 2x HNG ]] (100mL). Combine 100mM HEPES (10mL), 300mM NaCl (7.5mL), 20% glycerol (20mL) |
*1x HNG (10mL). Mix 5mL 2xHNG and 1 PI tablet. | *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. | *Lysis Buffer (10mL). Combine 5mL 1x HNG, 1 PI tablet, 1mL of 1% NP40, and 50µL of magnesium chloride. | ||
Latest revision as of 17:48, 31 July 2012
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.
