http://confocal-microscopy-list.275.s1.nabble.com/How-to-differentiate-plasma-membrane-vs-cytoplasm-in-Jurkat-lymphocytes-tp7586566p7586573.html
membrane fraction by FLIM. Membrane-bound fluorophore will have a shorter
and plasma membrane. See for reference "Refractive index sensing of green
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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
>
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> On 3/6/17, 2:47 PM, "Confocal Microscopy List on behalf of Swayne, Theresa
> C." <
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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<
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