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Immuno-staining for cell surface/membrane markers after permeabilisation


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#1 science noob

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Posted 03 December 2012 - 05:00 AM

What would happen if you permeabilised the cell membrane with detergent (e.g. Tween, Saponin, Triton X) and then stained those cells with a cell surface marker (e.g. mesenchymal or lymphocyte markers).

How would the staining turn out? cytoplasmic? reveals the pores created by detergent?

#2 bob1

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Posted 03 December 2012 - 11:13 AM

Depends on if the protein is also found inside the cell, not just on the surface. I don't think the pores would be visible, they will be too small to resolve.

#3 Curkobain

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Posted 05 December 2012 - 09:46 AM

When i use CD4 on T lymphocytes and then permeabilize (Th1-Th2, etc)...i loss the stain so i have to use more concentration of CD4...

I see the results via flow cytometry

#4 bob1

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Posted 05 December 2012 - 12:01 PM

@Curkobain, that's probably the effect of the detergents used to permeabilise, essentially this is creating a very stringent wash which removes the initial antibody binding.

#5 Tabaluga

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Posted 17 December 2012 - 05:45 AM

@science noob: I think that even if it's just a membrane protein and not normally expressed in the cell, the protein inside the Golgi could be stained if you permeabilize prior to staining (at least in FACS, in my experience, you'll get higher values then).

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