Oct 16, 2023

Public workspaceStrep pull down assay

DOI
dx.doi.org/10.17504/protocols.io.3byl4jmpolo5/v1
  • 1Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA
Minghao Chen
Open access
DOI: dx.doi.org/10.17504/protocols.io.3byl4jmpolo5/v1
Protocol Citation: Minghao Chen, Xuefeng Ren 2023. Strep pull down assay. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4jmpolo5/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: 82917
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s (ASAP) initiative
Grant ID: ASAP-000350
Abstract
Strep pull down assay for FIP200(NTD)-TSF WT or mutants were co-transfected with GST-ATG13(363-517) and/or ULK1(MIT)-MBP
Expression
Expression
2d 0h 40m
Transfect 10 ml 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.

Pull down
Pull down
2h 50m
Homogenize the pellets in 0.5 ml of lysis buffer/protease inhibitors/1% TritonX-100, and clarified after Centrifigation40000 x g , Duration00:15:00 .

15m
Incubate the lysate with 30 μl Strep-Tactin Sepharose resin (IBA-Lifesciences) at Temperature4 °C for Duration03:00:00

3h
Wash the beads four times, and eluted in 50 μl lysis buffer/4 mM desthiobiotin.

Mix 18 μl eluent with lithium dodecylsulfate (LDS)/BME buffer, heated at Temperature60 °C forDuration00:05:00 and subjected to SDS/PAGE gel. The gel was then stained with Coomassie brilliant blue G250.

5m