I have tried ammonium molybdate/ascorbic acid protocol and malachite green protocol with not much success. They are quite small quantities I am trying to quantify.Could anyone provide some guidance please? I'm not sure whether I'm digesting the lipid properly to release the phosphate.
First of all have you hypothesized, what would be the most probable phospholipids you are getting. If yes, then search for the right absorption maxima (in the UV range). You can get this from the literature or from its molecular structure. You may ask a friend or your colleague to help you out. Then simply calculate the area under the curve using a UV spectrophotometer out put. If you can get hold of a primary standard of your lipid (suspected), it will still be better.
Yes, I know I am quantifiying phosphatidylethanolamine. I'm sorry but I'm not quite sure I understand your way of doing it then. Could you explain it a bit better please?
I was digesting them in 260ul of 70% perchloric acid for 2 hours at 180 degrees...and then going onto ascorbic acid method. I have checked the article but it doesn't provide much detail. Would you be able to send me a more detailed protocol for the malachite green assay?I would be eternally thankful...
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