Jul 12, 2022
Expression and purification MBP-ATG9 Constructs
- 1Team Hurley
Protocol Citation: Adam Yokom 2022. Expression and purification MBP-ATG9 Constructs. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr6nkovmk/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 12, 2022
Last Modified: June 01, 2024
Protocol Integer ID: 66458
Keywords: ASAPCRN
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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
30m
Load supernatant flow thru gravity column onto washed Amylose resin
Rock supernatant with equilibrated resin for 01:00:00 at 4 °C
1h
Wash with 5 column volumes of lysis buffer (25 mM HEPES pH 7.5, 200 mM NaCl, 2 mM MgCl2, 1 mM TCEP, 5 mM EDTA, 10% Glycerol)
Elute with lysis buffer plus 40 mM Maltose for amylose resin
Concentrate elution and inject onto pre-equilibrated S200 10/30 column (25 mM HEPES pH 7.5, 200 mM NaCl, 2 mM MgCl2, 1 mM TCEP, 5 mM EDTA)
Pool peak fractions, concentrate, snap freeze, and store at -80C