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Tim Feinstein on
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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." <
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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.
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Theresa Swayne, Ph.D.
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