Oct 16, 2023

Public workspaceGST pull down assay

DOI
dx.doi.org/10.17504/protocols.io.36wgqj2xxvk5/v1
  • minghao chen1,
  • Xuefeng Ren1
  • 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.36wgqj2xxvk5/v1
Protocol Citation: minghao chen, Xuefeng Ren 2023. GST pull down assay. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqj2xxvk5/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: 82918
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s (ASAP) initiative
Grant ID: ASAP-000350
Abstract
GST pull down assay
Mix the GST tagged protein and fluorescent protein with 30 μl Glutathione Sepharose beads (Cytiva) at final 1 μM concentration in the buffer of 25 mM HEPES at pH 7.5, 150 mM NaCl, 1 mM MgCl2 and 1 mM TCEP. The total volume is 200 μl.
Rocking at Temperature4 °C DurationOvernight

Wash the beads four times, then eluted in 50 μl of buffer with 25 mM glutathione
Mix 18 μl eluent in lithium dodecylsulfate (LDS)/BME buffer and subjected to SDS/PAGE gel without heating samples. The gel was scanned at 488 or 550 nm in ChemiDoc MP imaging system (Bio-Rad).