Sep 06, 2022
Native-PAGE analysis of VCP hexamer
- Itika Saha1,
- F. Ulrich Hartl1,
- Mark S. Hipp2,3
- 1Department of Cellular Biochemistry, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany;
- 2Department of Biomedical Sciences of Cells and Systems, University Medical Center Groningen, University of Groningen, Antonius Deusinglaan, 1, 9713 AV Groningen, The Netherlands;
- 3School of Medicine and Health Sciences, Carl von Ossietzky University Oldenburg, 26129 Oldenburg, Germany
Protocol Citation: Itika Saha, F. Ulrich Hartl, Mark S. Hipp 2022. Native-PAGE analysis of VCP hexamer. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqjrzyvk5/v1
Manuscript citation:
The AAA+ chaperone VCP disaggregates Tau fibrils and generates aggregate seeds Itika Saha, Patricia Yuste-Checa, Miguel Da Silva Padilha, Qiang Guo, Roman Körner, Hauke Holthusen, Victoria A. Trinkaus, Irina Dudanova, Rubén Fernández-Busnadiego, Wolfgang Baumeister, David W. Sanders, Saurabh Gautam, Marc I. Diamond, F. Ulrich Hartl, Mark S. Hipp bioRxiv 2022.02.18.481043; doi: https://doi.org/10.1101/2022.02.18.481043
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: September 05, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 69590
Keywords: ASAPCRN, hek293 cell, tau repeat domain, eukaryotic cell, containing protein, hexamer formation, vcp, protein, cell, aggregates of tau repeat domain, valosin, page analysis of vcp, versions of vcp
Abstract
Valosin-containing protein (VCP) is a homo-hexameric AAA+ ATPase in eukaryotic cells. This protocol describes the analysis of myc-tagged versions of VCP transiently transfected in HEK293 cells (stably expressing and propagating aggregates of Tau repeat domain fused to YFP) for hexamer formation.
Troubleshooting
Plate 1.5x105 cells in 12-well plate.
Next day, transfect with plasmids expressing myc-tagged VCP variants (Saha et al. BioRxiv, 2022) using a standard transfection protocol.
Two days later, collect cells and lyse them in 50 µL 0.5% Triton X-100/PBS supplemented with protease inhibitor cocktail (Roche) and DNase for 1 h on ice.
1h
Centrifuge lysates at 10,000 x g for 2 min and collect supernatant.
2m
Determine protein concentration in the supernatant and normalize across all samples.
Add 2x native sample buffer (40 % glycerol, 240 mM Tris pH 6.8, 0.04 % bromophenol blue) to 40 µg lysate.
Run samples on a Native PAGE gel (e.g. Novex Value 4 to 12% Tris-glycine gels (Thermo)) in 20 mM Tris 200 mM Glycine buffer at pH 8.4.
1h
Transfer proteins to nitrocellulose membrane in standard Tris-glycine buffer, block in 5% low-fat dry milk for 1 h at room temperature (RT).
Note
NOTE: Nitrocellulose membranes produce less background than PVDF membranes with fluorescent secondary antibodies.
2h
Dilute anti-myc (9E10) and anti-VCP (1:2000, Novus Biologicals) primary antibodies together in blocking solution and incubate membrane overnight.
Next day, wash membrane 3 times with TBST and incubate with anti-mouse (LI-COR Biosciences Cat# 926-68070, RRID:AB_10956588; 1:10,000) and anti-rabbit (LI-COR Biosciences Cat# 926-32211, RRID:AB_621843; 1:10,000) fluorescent secondary antibodies for 2 h at RT.
2h
Wash membrane 3 times with TBST.
Detect fluorescent myc and VCP signal on a fluorescent imager.