Oct 17, 2023

Public workspaceExpression and purification for cryo-EM samples of FIP200NTD:ATG13(363-517)-ULK1(MIT) complexes

DOI
dx.doi.org/10.17504/protocols.io.e6nvwjxw7lmk/v1
  • 1Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA
Minghao Chen
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DOI: dx.doi.org/10.17504/protocols.io.e6nvwjxw7lmk/v1
Protocol Citation: Minghao Chen, Xuefeng Ren 2023. Expression and purification for cryo-EM samples of FIP200NTD:ATG13(363-517)-ULK1(MIT) complexes. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwjxw7lmk/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: June 05, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 82914
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP) initiative
Grant ID: ASAP-000350
Abstract
Expression and purification for cryo-EM samples of FIP200NTD:ATG13(363-517)-ULK1(MIT) complexes
Expression
Expression
2d 0h 40m
2d 0h 40m
Transfect HEK GNTi cells at concentration of 2 × 10^6 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 Duration00:30:00 at Temperature37 °C

30m
Add mixture to cells. Let cells grow for Duration48:00:00

2d
Harvest Cells Centrifigation500 rpm , Temperature4 °C , Duration00:10:00

10m
Wash pellet with cold PBS. Store pellet atTemperature-80 °C until purification.

Purification
Purification
2h 50m
2h 50m
Cell pellets were lysed at room temperature for 20 min with lysis buffer (25 mM HEPES pH 7.5, 200 mM NaCl, 2 mM MgCl2, 1 mM TCEP, 10% Glycerol) with 5 mM EDTA, 1% Triton X-100 and protease inhibitor cocktail (Thermo Scientific) for Duration00:15:00

15m
Clarify lysate for Centrifigation17000 rpm for Duration00:35:00 at Temperature4 °C .

35m
Wash strep-tactin resin (IBA Lifesciences, Germany) into lysis buffer (without Triton). Load clarified lysate onto resin
Rock supernatant with equilibrated Strep-Tactin Sepharose resin for Duration01:00:00 at Temperature4 °C
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
His6-TEV cleavage at Temperature4 °C DurationOvernight ,

1h
Concentrate elution and inject onto pre-equilibrated Superose 6 Increase 10/300 GL column (Cytiva) (25 mM HEPES pH 7.5, 150 mM NaCl, 1 mM MgCl2, 1 mM TCEP)
Pool peak fractions, concentrate, snap freeze, and store at Temperature-80 °C