GST-EEA1 Pulldown from Yeast: Difference between revisions

Latest revision as of 17:48, 31 July 2012

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

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.

@@ Line 1: / Line 1: @@
-enter protocol here
+== '''Materials''' ==
+*[[ 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.
+*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.