An alternative, effective substrate for erythrocyte acid phosphatase phenotype determinations |
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Authors: | B Budowle A M Gambel |
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Affiliation: | Forensic Science Research and Training Center, Laboratory Division, FBI Academy, Quantico, VA. |
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Abstract: | A method is described for obtaining nondiffusing, nonfading fluorogenic zymograms for erythrocyte acid phosphatase variants separated by isoelectric focusing. The synthetic substrate 4-trifluoromethylcoumarin phosphate was impregnated into cellulose diacetate membranes and air-dried overnight. After isoelectric focusing, the substrate overlay membrane was rehydrated in 0.05M citrate buffer, pH 4.0, lightly blotted, and overlayed on the gel. A 5- to 10-min incubation at 37 degrees C produced intensely fluorescing, light-blue bands on the diacetate membrane. Interaction of the trifluoromethyl group on the substrate with hydrophobic regions of the diacetate membrane impeded diffusion, yielding a permanent zymogram. There were no discrepancies in phenotype determinations using this method when compared with the 4-methylumbelliferyl phosphate assay approach. Further, an increased number of conclusive calls were obtained (91.8 versus 79.5% and 54.1 versus 34.9%) with this new assay when compared with the 4-methylumbelliferyl phosphate substrate on known liquid bloods and questioned dried bloodstains, respectively. |
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