Native-PAGE analysis of VCP hexamer
Itika Saha, F. Ulrich Hartl, Mark S. Hipp
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.
Steps
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.
Centrifuge lysates at 10,000 x g for 2 min and collect supernatant.
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.
Transfer proteins to nitrocellulose membrane in standard Tris-glycine buffer, block in 5% low-fat dry milk for 1 h at room temperature (RT).
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.
Wash membrane 3 times with TBST.
Detect fluorescent myc and VCP signal on a fluorescent imager.
