Hello everybody,
I wish to separate apoplast from the rest of the plant (i.e. intact cells) to see whether the compound I treat the plants with goes inside or stays outside. When I mentioned this to a colleague, she recommended apoplast wash method as in these papers
http://www.plantphysiol.org/content/179/4/1265.long
https://www.jove.com/video/52113/the-infiltration-centrifugation-technique-for-extraction-apoplastic
(it's both the same method)
However, I have probably not specified my goal exactly (probably I just said I want to separate apoplast and the cells). When I was reading these papers and thinking about it, I don't think this method is the right one for me, because I need to get rid of all of the apoplast and have pure cells (I don't care if some rupture), while this method aims rather for pure apoplast but not necessarily 100% of it, as I understand it.
Is here anybody with experience with this method?
Could you recommend some simple protocol to get rid of the apoplast and obtain pure cells?
Thank you.
Hi Tomas,
Good question. I think you assessed the situation correctly in that an apoplast wash method is not designed to collect 100% of the apoplast, but rather aims not to contaminate the recovered apoplast with cytosolic contents.
However, I think your purpose may be achievable by a modified version of this method if you can establish some controls. If you had an assayable compound that was only in the apoplast you could perform repeated AWF extractions from the same leaf until no more of this compound was detectable in the AWF. Presumable the leaf would then be rid of all its original apoplast. Of course the leaf cells with continue to interact and exchange compounds with the apoplast the whole time you are doing the procedure, but it might work.
I have never tried this though!
Cheers,
Brendan
Dear Brendan O'Leary
thanks for your reply.
I was thinking about using indigo carmine for the first round with subsequent round without it. But I'm afraid it would not be so sensitive as radioactivity (label on my compound).
I'm not sure if there is any compound that would be exclusively in the apoplast.
Hope this simple protocol can help you?
https://bio-protocol.org/e1691
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