Sep 23, 2023

Public workspaceImmunoprecipitation of NAP1-GFP

DOI
dx.doi.org/10.17504/protocols.io.5jyl8pmndg2w/v1
  • 1Sascha Martens lab, University of Vienna, Max Perutz Labs - Vienna
Elias Adriaenssens
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.5jyl8pmndg2w/v1
Protocol Citation: Elias Adriaenssens 2023. Immunoprecipitation of NAP1-GFP. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8pmndg2w/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 28, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 84128
Keywords: Immunoprecipitation, 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 immunoprecipitation of NAP1-GFP from HAP1 cells.
Attachments
Icon for 757-1925.pdf
757-1925.pdf
43KB
Materials
Materials

  • NAP1-EGFP (Addgene xxx)
  • Lipofectamine 2000 (Thermo Fisher)
  • SDS-PAGE gels (NP0322BOX, Thermo Fisher)
  • PageRuler Prestained protein marker (Thermo Fisher)
Lysis buffer
AB
KCl100 mM
MgCl22.5 mM
Tris-HCl pH 7.420 mM
NP-400.50%
Fixation solution

A
40% ethanol
10% acetic acid
50% dH2O


ReagentPierce™ Detergent Compatible Bradford Assay KitThermo FisherCatalog #23246
ReagentChromoTek GFP-Trap® AgaroseProteintechCatalog # gta-20

Immunoprecipitation
Immunoprecipitation
1h
1h
Transiently transfect FIP200 knockout HAP1 cells with pcDNA3.1 NAP1-EGFP (Addgene), or empty pcDNA3.1 vector as a negative control, using Lipofectamine 2000 (Thermo Fisher).
Note
This cell line was selected as FIP200 deletion results in TBK1 hyperactivation and thus increased NAP1 phosphorylation.

After 48 h, collect the cells by trypsinization and wash the cell pellet with PBS once. Then, lyse the cells in lysis buffer.
Lyse the samples for Duration00:20:00 TemperatureOn ice . Then, clear the cell lysates by centrifugation at Centrifigation20000 x g, 4°C, 00:10:00 .

30m
Centrifigation
Determine the protein concentrations of the cleared protein lysates with the Pierce Detergent Compatible Bradford Assay Kit (23246, Thermo Fisher).
For both samples, negative control and NAP1-EGFP lysates, incubate Amount12 mg of cell lysate DurationOvernight with Amount20 µL or GFP-Trap agarose beads (GTA-20, Chromotek).
20m
Incubation
Overnight
In the morning, wash the samples three times in lysis buffer. Then, resuspend the beads in protein loading dye, supplemented with 100 mM DTT. Finally, boil the samples for Duration00:05:00 at Temperature95 °C .

5m
Wash
Temperature
Load the samples on 4-12% SDS-PAGE gels (NP0322BOX, Thermo Fisher) with PageRuler Prestained protein marker (Thermo Fisher).
After the run, stain the SDS-PAGE gel with Coomassie, fix for Duration00:10:00 in fixation solution, and destain it DurationOvernight in dH2O.

15m
Overnight
Cut the band corresponding to NAP1-EGFP from the gel with a fresh scalpel and submit for mass spectrometry analysis.