Jul 20, 2022
Expression and purification Twin-STREP-FLAG tagged ATG13:ATG101 constructs
- Adam Yokom1,
- Xuefeng Ren1
- 1Team Hurley
Protocol Citation: Adam Yokom, Xuefeng Ren 2022. Expression and purification Twin-STREP-FLAG tagged ATG13:ATG101 constructs. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvmn4yng3p/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 11, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 66460
Keywords: ASAPCRN
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Abstract
Expression and purification Twin-STREP-FLAG tagged ATG13:ATG101 constructs
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) for 15 min
Clarify lysate for 17000 rpm, 4°C, 00:30:00
30m
Wash strep-tactin resin (IBA Lifesciences, Germany) into lysis buffer (without Triton). Load clarified lysate onto resin
Rock supernatant with equilibrated resin for 01:00:00 at 4 °C
Use glutathione resin for GST tagged proteins
Use amylose resin for MBP tagged proteins
Use Strep-Tactin Sepharose resin for Strep tagged proteins
1h
Wash with 5CV 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 4 mM desthiobiotin for STREP 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