I want to measure the different metabolites in plants. It would be really good if anyone could suggest the most suitable protocol.
Several kind of chromatographic methods can be used (LC-DAD, GC-FID, LC-MS...), it depends of appartus available for analyses in your lab.
Malate and OAA can be measured with enzyme-linked assays if you're really only interested in these two. e.g. using the MDH reaction and A340 - can be very sensitive e.g. with a fluorescence spec. so it depends whether you have ready access to HPLC or not. Important also to do measurements of recovery of known additions (spike) of OAA to the extract as this can be rather unstable in plant tissue.
I recommend you to have a look at the article tittled "A universal protocol for the combined isolation of metabolites, DNA, long RNAs, small RNAs, and proteins from plants and microorganisms" Valledor et al (2014). Maybe this can help you
We metabolite profile using a combination of GC-MS and LCMS. For malate and OAA the highly polar nature of these compounds means you need to use GCMS. You can use GC FID as suggested in an earlier post although you'll need to do some workup on ensuring you measure the correct compound first.
One of the criticial things you need to be aware of is the turnover rate of some metabolites. It can be seconds. When you sample your tissue make sure you flash freeze it with the minimal amount of time between cutting and dropping into liquid nitrogen. I would then freeze-dry your tissue. It should then be stable for the long term. You also want to use an extraction buffer which disrupts tissue and denatures enzymes all in one go, such as acidified acetonitrile. there are various reviews to guide you on this. Always ensure you have spiked your sample with an internal standard to account for extraction efficiency / ion suppression.
Feel free to email privately for more help.
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