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 11, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 66458
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only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind.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
Expression and purification from HEK cells of ATG13, ATG101 and FOLDON-ATG9A proteins
Expression
Expression
2d 12h 50m
2d 12h 50m
Transfect HEK GNTI cells at concentration of 2 × 106 cells/ml
Dilute PEI with Warm Hybridoma-SFM(1X)
In a separate tube, dilute DNA with Hybridoma-SFM(1X)
Add PEI to DNA dilution. Incubate mixture for 00:30:00 at 37 °C
30m
Add mixture to cells. Let cells grow for 48:00:00
2d
Harvest Cells 500 rpm, 4°C, 00:10:00
10m
Wash pellet with cold PBS. Store pellet at -80C until purification.
Purification
Purification
2d 12h 50m
2d 12h 50m
Resuspended pellet in lysis buffer (25 mM HEPES pH 7.5, 200 mM NaCl, 2 mM MgCl2, 1 mM TCEP, 5 mM EDTA, 10% Glycerol) with 1% Triton X-100 and protease inhibitor cocktail (Thermo Scientific, Waltham, MA)
Wash Amylose resin with lysis buffer, ~2mL/1 Liter of cells
Clarify lysate by centrifugation 17000 rpm, 4°C, 00:30:00