I have some plants with altered enzyme expression and I'm interested in following compounds:
- flavins (mainly FAD and FMN, not sure whether it's possible to discriminate OR to include those bound in proteins?)
- NAD(P)+ and NAD(P)H
- ATP, mainly extracellular if possible
Is anybody here familiar with methods for extraction, purification and determination of such compounds in plants (not food)?
Kits are available from many suppliers for determination of ATP and NAD(P)/(NAD(P)H concentrations in an extract.
Hi Hanna-Maija, thanks for your helpful answer.
You remove the 250ul of methanol/methylene chloride (CH2Cl2, right?) and use 100ul from that, right? To the pellet only (not pellet + 150ul of methanol/methylene chloride) you add the additional 250ul and afterwards you mix 100ul from both, right?
Unfortunately we have only UV-VIS detector, so I hope I'll be able to see something at all.
I found the second paper you mentioned. However, they use some weird buffer and I'm not sure of the composition. They write it's 0.1M ammonium acetate with 10% TCA pH 6.1, which doesn't make much sense. Mainly it's out of the buffering range for acetate (pKa 4.76) and in the end they adjust the pH of extract to 6.3, which would not be such a big change. I've looked up the original paper by group from Gatersleben, but that one is not much clearer. And nobody seems to care to reply to e-mail.
and the methanol/methylene chloride is e.g. 90ml of methanol mixed with 100ml of methylene dichloride, right?
Hi Hanna-Maija once again. I have few more questions.
The second extraction buffer is pH 5.5 like the mobile phase A?
You wrote there is no real pellet and only some green phase. I had this green phase only in one sample (I tried 2 samples to see how it works and whether I'll get anything), while in both was normal pellet. Could it be insifuciently crushed? We have such small sticks for crushing material in the microtube, so maybe it was not enough?
And last, so far, but the most important - how do you quantify the flavins as per material? What I mean is, that you extract with two different solutions and you always take only part of it, so you cannot really say how much flavins there was in the plant material in the beginning, can you?
And one more thing actually. When I ran my standard on the fluorescent detector, I got at least six peaks (the FMN was only 80% purity), maybe even nine. Three of them are obvious (and confirmed by the standards - FAD, FMN, riboflavin), but what could be the other three/six?
Thank you very much!
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