Sep 23, 2023

Public workspaceGFP pull down assay

DOI
dx.doi.org/10.17504/protocols.io.e6nvwd6x2lmk/v1
  • 1Sascha Martens lab, University of Vienna, Max Perutz Labs - Vienna
Elias Adriaenssens
Open access
DOI: dx.doi.org/10.17504/protocols.io.e6nvwd6x2lmk/v1
Protocol Citation: Elias Adriaenssens 2023. GFP pull down assay. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwd6x2lmk/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 27, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 84086
Keywords: GFP pull down assay, ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-000350
Marie Skłodowska-Curie MSCA Postdoctoral fellowship
Grant ID: 101062916
Abstract
This protocol describes GFP pull down assay.
Attachments
754-1922.pdf
754-1922.pdf
43KB
Materials
Materials

  • GFP-Trap agarose beads (Chromotek)
  • dH2O
  • Protein Loading dye

Bead assay buffer
AB
Tris-HCl pH 7.425 mM
NaCl150 mM
DTT1 mM
GFP pull down assay
GFP pull down assay
2h 5m
Mix GFP-tagged TBK1 with Amount20 µL of equilibrated GFP-Trap agarose beads (Chromotek) at a final concentration of Concentration1 micromolar (µM) . Make sure to wash beads 2x in dH2O before washing with bead assay buffer to equilibrate the beads adding the protein to the beads.
To this end, wash Amount20 µL of beads twice with dH2O and equilibrate with bead assay buffer.
Wash
Resuspend the beads in Amount40 µL bead assay buffer, to this add GFP-TBK1 at a final concentration of Concentration5 micromolar (µM) .
Pipetting
Incubate the beads with GFP-TBK1 for Duration01:00:00 at Temperature4 °C at a horizontal tube roller.
1h
Incubation
Wash the beads three times to remove unbound GFP-tagged bait protein.
Wash
Prepare protein master mixes with prey protein in bead assay buffer at the following concentrations:
  • mCherry-OPTN (1 µM),
  • mCherry-NDP52 (1 µM),
  • GST-NAP1 (1-10 µM).
Add the protein master mixes to the beads and incubate for Duration01:00:00 at Temperature4 °C at a horizontal tube roller.
1h
Incubation
Pipetting
Wash the beads three times to remove unbound proteins, remove any supernatant from the beads and resuspend the beads in Amount60 µL of 1x Protein Loading dye, and heat-inactivate at Temperature95 °C for Duration00:05:00 .
5m
Wash
Temperature
Analyze the samples by SDS-PAGE and Coomassie staining as described above.
Analyze