Authors: Aubin Penna
This procedure describes how to assess the native quaternary structure of membrane proteins by PAGE using mild solubilization in the non-dissociative detergent perfluoro-octanoic acid (PFO). The number of subunits is deduced from the molecular mass of the preserved homo-multimeric protein complex. The PFO-PAGE technique was first described by Ramjeesingh, M et al. (1999) and applied later to determine the subunit stoichiometry of other channels (e.g. Kedei, N. et al. (2001) and in the present study to Orai and P2X2 expressed in Drosophila S2 cells or human embryonic kidney HEK293 cells).
- Rinse the cells twice with ice-cold phosphate-buffered saline (PBS).
- Harvest the cell pellet in ice-cold PBS supplemented with protease inhibitors and sonicate. Alternatively, solubilize the cells in the presence of 1% NP-40, a mild detergent diluted in PBS, for 20 min at 4°C under agitation and centrifuged at 16,000 × g for 10 min to remove cellular debris.
- Measure the protein content by a small-volume micromethod using the Bio-Rad DC protein assay.
- Mix 30-40 µg of lysates at 2 µg/µl of protein with doubly concentrated PFO sample buffer (100 mM Tris base, 2-8% NaPFO (Oakwood Products Inc), 20% glycerol, and 0.005% bromophenol blue, pH adjusted at 8.0 with NaOH) and 25 mM DTT.
- Incubate at room temperature for 25 min, vortex briefly, and centrifuge 5 min at 10,000 × g.
- Load your samples at room temperature on a 4-12% precast gradient Novex Tris-Glycine gel without SDS (Invitrogen) along with molecular weight standards resuspended in PFO sample buffer (high-molecular-mass rainbow marker kit (GE Healthcare), cross-linked Albumin and phosphorylase b (Sigma)).
- Perform electrophoresis at 140 V with a running buffer (25 mM Tris, 192 mM glycine, 0.5% NaPFO, pH 8.5, adjusted with NaOH) precooled to 4°C. During the run, the electrophoresis box is kept at 4°C.
- Transfer the proteins to a nitrocellulose membrane for 1 hr in a transfer buffer containing 25 mM Tris, 192 mM glycine and 20% (v/v) methanol.
- Analyze the protein band sizes either by Amido black staining (Sigma) for the molecular weight standards or by standard immunoblotting.
- Ramjeesingh, M., Huan, L. J., Garami, E. & Bear, C. E. Novel method for evaluation of the oligomeric structure of membrane proteins. Biochem. J. 342 ( Pt 1), 119-23 (1999).
- Kedei, N. et al. Analysis of the native quaternary structure of vanilloid receptor 1. J. Biol. Chem. 276, 28613-9 (2001).
The CRAC channel consists of a tetramer formed by Stim-induced dimerization of Orai dimers, Aubin Penna, Angelo Demuro, Andriy V. Yeromin, Shenyuan L. Zhang, Olga Safrina, Ian Parker, and Michael D. Cahalan, Nature 456 (7218) 116 - 120 06/11/2008 doi:10.1038/nature07338
Aubin Penna, Department of Physiology and Biophysics, University of California, Irvine, California, USA 92697
Correspondence to: Aubin Penna ([email protected])
Source: Protocol Exchange (2008) doi:10.1038/nprot.2008.210. Originally published online 7 October 2008.