Jul 20, 2022
MBP Pulldown Assay of ATG9A Truncations
Forked from WIPI2d Coprecipitation Assay
- Adam Yokom1,
- Xuefeng Ren1
- 1Team Hurley
Protocol Citation: Adam Yokom, Xuefeng Ren 2022. MBP Pulldown Assay of ATG9A Truncations. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwk7xwvmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: July 08, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 66238
Keywords: ASAPCRN
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Abstract
MBP Pulldown Assay of ATG9A Truncations
Equilibrate 30 μl of Amylose resin (New England Biolabs, Ipswich, MA). Add >500mL of wash buffer (25 mM HEPES pH 7.5, 150mM NaCl, 1mM MgCl2, 1mM TCEP). Slow spin to pellet resin. ~1000rpm for 1 minute should be good. Repeat X3
Add recombinant MBP protien (~1 uM) and ATG13:ATG101 dimer (~3 uM) to Amylose resin
Incubate overnight at 4C
Wash resin 4x with wash buffer (25 mM HEPES pH 7.5, 150mM NaCl, 1mM MgCl2, 1mM TCEP)
Elute samples in 50 uL buffer + 50 mM Maltose
Mix eluted samples with lithium dodecylsulfate (LDS)/BME buffer. Heat at 60C for 5 min and run on SDS/PAGE gel
Quantify using Fiji ImageJ2