I have come across an ELISA to measure RA levels in serum/plasma, however I am not sure if this sensitive enough to determine RA concentrations in cell lysates (0.625-10 ng/mL). I was hoping someone could provide insight into this product? Likewise, if anyone has measured intracellular RA using HPLC, what concentration of RA/cell did you obtain.
https://www.mybiosource.com/prods/ELISA-Kit/Human/Retinoic-Acid/datasheet.php?products_id=705877
Thank you,
Tyler
Dear Sir. Concerning your issue about the determination of intracellular retinoic acid (RA) concentrations in the cytosol of cultured cells. Prior to cytosol preparation, the epithelium was discharged from adhering blood and muscle by scissors and weighed. Then the biopsy was homogenised with an electric micro-homogeniser in homogenising bu€er (10 mM KCl, 10 mM K2HPO4, 0.5 mM EDTA, 0.5 mM phenylmethylsulfonyl ¯uoride, 6000 U aprotinin, pH 7.4) and centrifuged at 150 000 ´g for 1 h. The protein content of the supernatant was adjusted to 1±2 mg/ml with homogenising bu€er used as cytosol. The cytosol was incubated with 50 nM [3H]RA for 15 min at ambient temperature. Excess [3H]RA was removed by dextran coated charcoal. Speci®c binding was separated from non-speci®c binding by discontinuous polyacrylamide gel electro- phoreses (slide size: 16 ´ 13 ´ 0.1 cm; apparatus: Protean II xi, BioRad, MuÈ nchen, Germany; 12% acrylamide in the separation gel) in the cold. For the determination of total CRABP the cytosol was incubated with 0.5 mM PCMBS for 1.5 h at ambient temperature to displace the retinoic acid already bound to CRABP. The ability to bind RA was restored by incubation with 5 mM dithiothreitol for 15 min. In this cytosol all the CRABP is present in the apo form. Incubation with [3H]RA leads to total CRABP. I think the following below links and the attached file may help you in your analysis:
http://www.axxora.com/fileadmin/files/manual/apo-54n-038_manual_v02_081202.pdf
https://www.mybiosource.com/prods/ELISA-Kit/Human/Retinoic-Acid/datasheet.php?products_id=705877
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3003897/
http://journals.tubitak.gov.tr/veterinary/issues/vet-10-34-3/vet-34-3-9-0904-15.pdf
Thanks
Retinoic Acid is a form of Vitamin A. You can the use Antimony Trichloride which gives a transient 'blue' color but your sample has to be totally dehydrated since water quenches the reaction.
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