http://confocal-microscopy-list.275.s1.nabble.com/How-to-differentiate-plasma-membrane-vs-cytoplasm-in-Jurkat-lymphocytes-tp7586566p7586581.html
associated protein. They published that it was external and a much sought
after ECM receptor. Turned out it was internal and the retraction was
forthcoming and quite embarrassing. Many other examples of this. IMHO it
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>
> The cytosolic fraction of a protein can be differentiated from the
> membrane fraction by FLIM. Membrane-bound fluorophore will have a shorter
> lifetime; this is based on difference between refractive indices of cytosol
> and plasma membrane. See for reference "Refractive index sensing of green
> fluorescent proteins in living cells using fluorescence lifetime imaging
> microscopy" Biophysical Journal (2008), 94 (8), L67-L69
> Best regards
>
> On Mon, Mar 6, 2017 at 3:31 PM, Feinstein, Timothy N <
[hidden email]>
> wrote:
>
> > *****
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> >
> > If you have to work with fixed cells, TIRF might be a good way to go.
> > Cytosolic proteins would get brighter as you adjust the TIRF angle from
> > more stringency to less while PM proteins would not. If you are working
> > with live cells, FRAP or FLIP would tell you whether they diffuse like
> > membrane-bound proteins or soluble proteins. Membrane mobility would go
> > down significantly if you decrease the temperature a little.
> >
> > Tim
> >
> > Timothy Feinstein, Ph.D.
> > Research Scientist
> > University of Pittsburgh Department of Developmental Biology
> >
> >
> > On 3/6/17, 2:47 PM, "Confocal Microscopy List on behalf of Swayne,
> Theresa
> > C." <
[hidden email] on behalf of
>
[hidden email]>
> > wrote:
> >
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> >
> >
> > Dear fellow confocalists,
> >
> > One of my users would like to determine whether an epitope-tagged
> > protein, expressed in Jurkat cells, is localized to the plasma membrane
> or
> > the cytosol. These cells have only a thin layer of cytoplasm between the
> > nucleus and PM.
> >
> > It seems to me that at minimum we need a good plasma membrane marker,
> > and control proteins known to be in cytosol and plasma membrane
> > respectively. Then I’m thinking we could do scanning confocal with high
> NA
> > lens, and profile analysis or overlap analysis to measure the degree of
> > coincidence between the membrane marker and the tagged protein. Does
> that
> > make sense as a strategy?
> >
> > If so, do folks have a preferred membrane marker for this cell type?
> > I’ve seen things like CellMask and PKH26, but the images I’ve seen online
> > and in other cell types don’t show “pure” plasma membrane labeling.
> >
> > Thanks in advance for any suggestions.
> >
> >
> > ------------------------------------
> > Theresa Swayne, Ph.D.
> > Manager
> > Confocal and Specialized Microscopy Shared Resource<
https://na01.
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> >
> > Herbert Irving Comprehensive Cancer Center
> > Columbia University Medical Center
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> > Phone: 212-851-4613
> >
[hidden email]<mailto:
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> >
> >
> >
> >
>
>
> --
> Artem Pliss
> Research Assistant Professor
> 432 Natural Sciences Complex
> Institute for Lasers, Photonics and Biophotonics
> State University of New York, University at Buffalo
> Buffalo, NY 14260 - 3000
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> Fax: (716) 645 6945
>