Hi people, can anybody recommend conditions (buffers, protocols, activity tests) for an assay with isolated chloroplasts. I want to study isoprenoid formation and need the basic machinery of the pastid to work.
Here is what I do: I isolate chloroplasts by filtering homogenized Arabidopsis leaves and concentrating them by centrifugation. I use buffers containing Sorbitol, HEPES and MgCl2, but I suspect there is a lot missing that chloroplasts need to be happy :)
Phosphate? ADP? Protease inhibitors? And how can I be sure they are still alive and well?
I'd be happy to hear from you! Any literature reference is welcome!
I use the method for the preparation of pea and spinach chloroplasts of Siegenthaler and Dumont 1990 Plant Cell Physiology 31: 1101-1108 . To check that your chloroplasts are good to go the best way is oxygen evolution using an oxygen electrode
Check out these papers:
ZE /VDE assays in arabidopsis and in vitro:
1. Bouvier F, D’Harlingue A, Hugueney P, Marin E, Marion-Poll A, Camara B. Xanthophyll biosynthesis. Cloning, expression, functional reconstitution, and regulation of beta-cyclohexenyl carotenoid epoxidase from pepper (Capsicum annuum). J Biol Chem. 1996;271(46):28861–7.
1. Kuczyńska P, Latowski D, Niczyporuk S, et al. Zeaxanthin epoxidation - an in vitro approach. Acta Biochim Pol. 2012;59(1):105–7.
lettuce:
1. Siefermann D, Yamamoto HY. NADPH and oxygen-dependent epoxidation of zeaxanthin in isolated chloroplasts. Biochem Biophys Res Commun. 1975;62(2):456–61.
Do you need intact chloroplast? And do you need photosynthetically active chloroplasts? If the answer on the 1st question is yes, so it is almost impossible to isolate intact chloroplast from Arabidopsis. But we managed to isolated quite good chloroplasts from Arabidopsis by grinding leaves with subsequent spin in percoll gradient. If you are interested I can send you the method with more details. Also, intactness of chloroplast you can check by ferrocyanide addition like it was described here http://jxb.oxfordjournals.org/content/61/13/3577.full
Thank you for your replies. I tried the method described in this paper
Seigneurin-Berny, D., Salvi, D., Joyard, J., & Rolland, N. (2008). Purification of intact chloroplasts from Arabidopsis and spinach leaves by isopycnic centrifugation. Current Protocols in Cell Biology / Editorial Board, Juan S. Bonifacino ... [et Al.], Chapter 3, Unit 3.30. doi:10.1002/0471143030.cb0330s40
But I had no success. I will switch to spinach leaves because of better availability and more reports of high activity of isolated chloroplasts...
Did you try the method described in in this paper?
http://www.ncbi.nlm.nih.gov/pubmed/12372603
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