MBP Pulldown Assay of ATG9A Truncations
Xuefeng Ren, Adam Yokom
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Abstract
MBP Pulldown Assay of ATG9A Truncations
Steps
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
