2015
DOI: 10.1016/j.bpj.2015.04.021
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In Situ Quantification of Protein Binding to the Plasma Membrane

Abstract: This study presents a fluorescence-based assay that allows for direct measurement of protein binding to the plasma membrane inside living cells. An axial scan through the cell generates a fluorescence intensity profile that is analyzed to determine the membrane-bound and cytoplasmic concentrations of a peripheral membrane protein labeled by the enhanced green fluorescent protein (EGFP). The membrane binding curve is constructed by mapping those concentrations for a population of cells with a wide range of prot… Show more

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Cited by 17 publications
(26 citation statements)
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“…2B and D), which provides information about the axial distribution of the Gag-eYFP at the scan location (61). Fitting of the z-scan intensity profile separates the fluorescence contributions from the cytoplasmic and the PM-bound protein populations (35,36). For a cell without Gag-eYFP at the PM (Fig.…”
Section: Methodsmentioning
confidence: 99%
See 1 more Smart Citation
“…2B and D), which provides information about the axial distribution of the Gag-eYFP at the scan location (61). Fitting of the z-scan intensity profile separates the fluorescence contributions from the cytoplasmic and the PM-bound protein populations (35,36). For a cell without Gag-eYFP at the PM (Fig.…”
Section: Methodsmentioning
confidence: 99%
“…The dual-color z-scan technique uses vertical motion (a so-called z-scan) of the laser focus to measure fluorescent intensity versus cell height at individual locations within a cell. A deconvolution model was applied to the fluorescent intensity traces resulting from each z-scan (35,36). The model was used to compare, within constraints imposed by the deconvolution, the vertical distribution of the mCherry fluorescence to that of Gag-eYFP.…”
mentioning
confidence: 99%
“…Increased PM affinity under low salt conditions has been reported in the case of Rouse sarcoma virus MA (42) and HIV-1 MA (25). Interestingly, the dissociation constant estimated by a previous SC z-scan study of HTLV-1 MA was ~200 nM (11), which differs from that of HIV-1 MA by nearly two orders of magnitude. These results serve to emphasize the striking diversity among retroviruses, particularly the orthoretroviruses, despite the relative conservation of retroviral MA domain structure.…”
Section: Discussionmentioning
confidence: 85%
“…The radially integrated PSF, provides the foundation for modeling z-scan intensity profiles (12). The normalized observation volume is defined by definite integration of the RIPSF function (11),…”
Section: Methodsmentioning
confidence: 99%
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