We have been using 0.1% saponin to permeabilize HEK-293T cells in order to introduce membrane impermeant dyes for FRET studies. Typically we discard the cells immediately after the experiment but recently, I left the cells overnight at 37C in the "loading buffer" (i.e. saponin plus fluorescent dyes) and I was quite surprised the next day to see the cells doing well and the dyes trapped inside the cell. This would suggest that the saponin has been inactivated somehow or that the cells figured out a way to live even in its presence. Is there any precedence for this?
Yes saponin effect is reversible. Therefore, all permeabilization protocols such as that of the Cytofix/Cytoperm kit from B&D call for saponin presence in all steps where membrane permeabilization is needed...
Yes saponin effect is reversible. Therefore, all permeabilization protocols such as that of the Cytofix/Cytoperm kit from B&D call for saponin presence in all steps where membrane permeabilization is needed...
wow, that's interesting.
we use saponin for our immunostainings, in a comparably low conc. for only 2 min to permeabilize the membrane, and all the rest of the steps are WITHOUT saponin and we get normal staining... Though, we work on already fixed cells. Maybe, that's the reason?
I have used digitonin for cell permeabilization and subsequent loading cells with antibodies. Permeabilization was reversible for neuronal PC12 cells but not for fibroblasts 3T3.
Look at this!! I think this short paper by Medipalli and cols answers quite well your question
http://iopscience.iop.org/0957-4484/24/20/205101/article
I am trying to do the same followed by FACS
Regards,
Josema
I was wondering if the saponin treatment for permeabilization can be used for labelling endogenous protein by a secondary-conjugated antibody and if the analysis by microscope in vivo is possible.
Thank you
Hi all ... James, Yaroslav, Irina and all :-) .. I am just starting with live-permeabilisation of PM setup. And I am new to saponins. What in your experience would be the some low concentration of Saponin (especially some low - just nearly working concentration) and what is actually the best solvent to make stocks of Saponin? (In what solvent should I dissolve it and what I may expect from its solublity)?
I do have available Sigma Saponin 8047-15-2 .. (And some others) And I would fetch anything, which would allow me just to somewhat permeabilize PM, but would let endomembranes intact. Should I try Digitonin? Is it better characterised? Better for permeabilization?
Thank You.
So, can Saponin, at 0.1%, can induce big enough and reversible pores for antibodies to enter live cells?
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