We are working on extraction of apoplast from 2-week-old Arabidopsis seedlings.
We use vacuum infiltration-centrifugation technique.
Unfortunately, we have not been able to isolate apoplast without cytosolic contamination!
When we run SDS-PAGE, Rubisco bands are found!
Do you have any suggestions for improving our apoplast isolation without cytosolic contamination?
Does anyone have any successful experience with apoplast isolation from Arabidopsis?
Thanks
Dear Tayebeh Abedi ,
Apoplast isolation from plant tissues is a challenging task, and it can be difficult to achieve complete separation from the cytosol.
Here are a few suggestions that may help improve your apoplast isolation:
It's important to keep in mind that even with the best isolation methods, it's difficult to achieve complete separation of the apoplast from the cytosol, and some cytosolic contamination is to be expected.
It's also worth trying to find literature about the apoplast isolation from Arabidopsis, as some researchers may have published their methods and results and could be a good source of information.
Freeze/thawing of samples and centrifugation, maybe.
Note that Rubisco is chloroplastic and not cytosolic. This protein represents 50% of total leaf soluble proteins. Difficult to totally exclude contamination by Rubisco
Dear Nicolò M. Villa
I really thank you for your response and the tips you mentioned.
But I think increasing the vacuum pressure and centrifugation speed would result in more cytosolic contamination.
Dear Claude Alban
I really thank you for your answer.
I use fresh plant not freezed one.
I know that Rubisco is a chloroplastic protein. I mean when I have Rubisco in my apoplastic extract it shows that I have some contamination from inside the cells what is problematic in my experiments.
Hey Tayebeh,
I hope you are doing fine!!
I am currently doing apoplast extractions from tobacco leaves, and we do not see the rubisco protein inside our samples. I think the main problem could be the damage of the seedlings itself during the extraction. You could try to reduce the centrifugation speed during the extraction, which will cause less damage to the seedlings.
Also you could try to give the seedlings less pressure during the vacuum infiltration. This should keep the seedlings intact.
If this doesn't work, you might just try to purify your protein of interest with Cation Exchange. This will only work if the iso electric point is different of course.
May I ask what you try to purify from the apoplast?
Pieter Nibbering
Hi Pieter,
I was really excited to hear from you and I appreciate your tips.
I agree that the problem we encountered with the apoplast isolation was caused by seedling damage.
To address this issue, I have tried to apply less pressure during the vacuum infiltration and to reduce the centrifugation speed during the extraction (down to 800 *g). However, since I am working with two-week-old Arabidopsis plants that are cultured in half MS medium, the seedlings are very fragile and easily damaged during apoplast extraction.
My current research is focused on examining the presence of glucosidase enzyme in the apoplast under stress conditions that also exist inside cells. As a result, the cytosolic contamination of the apoplast extract is interfering with our research goals.
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