Abstract
The subunit number and stoichiometry of membrane-bound proteins are difficult to determine without disrupting their membrane environment. Here we describe a single-molecule technique for counting subunits of proteins in live cell membranes by observing bleaching steps of GFP fused to a protein of interest. After testing the method with proteins of known stoichiometry expressed in Xenopus laevis oocytes, we resolved the composition of NMDA receptors composed of NR1 and NR3 subunits.
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Acknowledgements
We thank E.C. Young for the X-fA4 channel DNA. This work was supported by postdoctoral fellowships from the Deutsche Forschungsgesellschaft and the American Heart Association to M.H.U., and by a grant from the US National Institutes of Health.
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Supplementary information
Supplementary Fig. 1
Example traces for NR1+NR3B receptor bleaching steps. (PDF 91 kb)
Supplementary Video 1
Photobleaching of GFP fused to a tetrameric CNG channel expressed in the membrane of a Xenopus laevis oocyte. (MOV 1251 kb)
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Ulbrich, M., Isacoff, E. Subunit counting in membrane-bound proteins. Nat Methods 4, 319–321 (2007). https://doi.org/10.1038/nmeth1024
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DOI: https://doi.org/10.1038/nmeth1024
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