WIPI2d Coprecipitation Assay
lmstrong
Published: 2021-08-23 DOI: 10.17504/protocols.io.bxkspkwe
Abstract
Pulldown assay of WIPI2d
Steps
1.
Equilibrate 10uL of GST resin to pulldown buffer. To do this, pipette 40uL of slurry (so 20uL of resin) into a 1.7uL eppy. Add >500mL of wash buffer. Slow spin to pellet resin. ~1000rpm for 1 minute should be good. Repeat X3
2.
Add 10 μM purified WIPI2d and either with 20 μM of GST or GST-ATG16L1(207-230). Add buffer (25mM HEPES pH 7.4, 150mM NaCl, 1mM TCEP) to final volume of 150uL.
3.
Let rock at 4C for 2 hour. Wash x3
4.
Add 10uL of SDS-Loading dye to beads
5.
Run beads on SDS-PAGE gel and stain with Coomassie.
