Dear Colleagues,
I am looking for a method that could be used to measure FAD concentration (Flavin adenine dinucleotide) in biological samples, such as nectar or plant bleeding sap. Hope any experienced colleague could provide a detailed protocol, using HPLC or not.
I also wonder whether there is a method I can use to isolated FAD binding proteins from my samples such as floral nectar.
Any useful clues will be appreciated.
Zha
Dear Sir. Concerning your issue about the quantification of FAD (Flavin adenine dinucleotide) in biological samples (nectar or plant bleeding sap). Flavin cofactors, in particular flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), are critical for cellular redox reactions and sense light in naturally occurring photoreceptors and optogenetic tools. Here, three procedures for extraction of free and noncovalently protein-bound flavins and verified extraction using fluorescence spectroscopy . For separation, two CE methods with different BGEs were established, and detection was performed by LED-induced fluorescence with limit of detections (LODs 0.5-3.8 nM). We found that riboflavin (RF), FMN, and FAD contents varied significantly between cell lines. RF (3.1-14 amol/cell) and FAD (2.2-17.0 amol/cell) were the predominant flavins, while FMN (0.46-3.4 amol/cell) was found at markedly lower levels. Observed flavin contents agree with those previously extracted from mammalian tissues, yet reduced forms of RF were detected that were not described previously. Quantification of flavins in mammalian cell lines will allow a better understanding of cellular redox reactions and optogenetic tools. I think the following below links and the attached file may help you in your analysis:
http://www.jbc.org/content/223/1/559.full.pdf
https://www.ncbi.nlm.nih.gov/pubmed/25488801
http://www.abcam.com/flavin-adenine-dinucleotide-fad-assay-kit-ab204710.html
http://www.nature.com/pr/journal/v67/n3/pdf/pr201055a.pdf
Thanks
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