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
Disclaimer
DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK
The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with protocols.io, can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services.
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