TY - JOUR
T1 - An alternative, effective substrate for erythrocyte acid phosphatase phenotype determinations
AU - Budowle, B.
AU - Gambel, A. M.
PY - 1988
Y1 - 1988
N2 - A method is described for obtaining nondiffusing, nonfading fluorogenic zymograms for erythrocyte acid phosphatase variants separated 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°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 discepancies in phenotype determinations using this method when compared with the 4-methyl-umbelliferyl 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 blood-stains, respectively.
AB - A method is described for obtaining nondiffusing, nonfading fluorogenic zymograms for erythrocyte acid phosphatase variants separated 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°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 discepancies in phenotype determinations using this method when compared with the 4-methyl-umbelliferyl 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 blood-stains, respectively.
UR - http://www.scopus.com/inward/record.url?scp=0023813888&partnerID=8YFLogxK
U2 - 10.1520/jfs12514j
DO - 10.1520/jfs12514j
M3 - Article
C2 - 3171508
AN - SCOPUS:0023813888
SN - 0022-1198
VL - 33
SP - 915
EP - 920
JO - Journal of forensic sciences
JF - Journal of forensic sciences
IS - 4
ER -