Fluorometric determination of nanogram quantities of protein in small samples: application to calcium-transport adenosine triphosphatase.
A fluorometric micro protein assay based on fluorescamine-labelling of homogeneous proteins in solution has been developed which is capable of accurately quantitating as little as 25 ng protein at a concentration of 1.25 micrograms/ml. This micro assay uses a flow-through HPLC fluorescence detector. Typical micro assays measuring bovine serum albumin standards (0-25 mg/l) yielded linear regression coefficients of r = 0.999. Assays of purified Ca2+-ATPase solutions determined by the micro fluorescamine procedure correlated well with measurements made using the deoxycholate-TCA-precipitation modification of the Lowry assay: 1.0 microgram ATPase by Lowry method = 1.1 microgram protein by fluorescamine microassay (when both procedures were standardized with bovine serum albumin) (r = 0.995). The assay proposed offers a 100-fold increase in sensitivity, compared to the Lowry procedure.
D000252:Calcium-Transporting ATPases / Q000097:blood*; D002851:Chromatography, High Pressure Liquid; D004910:Erythrocyte Membrane / Q000201:enzymology; D005450:Fluorescamine; D006801:Humans; D008832:Microchemistry / Q000379:methods; D013141:Spiro Compounds
D013141:Spiro Compounds; D005450:Fluorescamine; D000252:Calcium-Transporting ATPases