Phosphoglucomutase‐1 subtyping of human bloodstains on ultrathin‐layer polyacrylamide gels

Budowle, Bruce

doi:10.1002/elps.1150050309

Cited by 26 publications

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“…Isoelectric focusing (IEF) has become a valuable technique in the field of forensic serology, and the use of ultrathin gel is making it even more powerful (335). IEF has been used to type esterase D (ESD) (336)(337)(338), group specific component (Gc) (339-343, 335), erythrocyte acid phosphatase (ACPi) (344), transferrin (Tf) (345), and phosphoglucomutase (PGM) (346)(347)(348)(349)(350)(351)(352)335). The value of ultrathin polyacrylamide gels for the simultaneous separation of PGM and EAP by isoelectric focusing of human red cell lysates and bloodstain extracts has been investigated (353).…”

Section: Forensic Biochemistrymentioning

confidence: 99%

Forensic science

Brettell¹,

Saferstein²

1985

Anal. Chem.

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Section: Forensic Biochemistrymentioning

confidence: 99%

Forensic science

Brettell¹,

Saferstein²

1985

Anal. Chem.

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Rapid electrofocusing of erythrocyte acid phosphatase

Budowle

1984

Electrophoresis

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A routine method is described for obtaining reproducible erythrocyte acid phosphatase patterns by ultrathin-layer polyacrylamide gel isoelectric focusing. Electrofocusing of EAP on 0.2 mm thick gels took 30 and 22 min on the Ultrophor and Cold Focus Apparatus, respectively, when the distance separating the electrodes was 5.4 cm. This method, which reduces the cost for focusing EAP, can be ideal for many laboratories interested in EAP phenotyping.Erythrocyte acid phosphatase (EAP) is governed by 3 autosoma1 codominant alleles designated A, B and C [ 11. Conventional electrophoresis has been the method of choice for separating the EAP variants [ 1-61. Following electrophoresis, the EAP phenotypes are detected by enzymatic activity. However, the determination of the C, CB and B phenotypes relies on a subjective comparison of band intensities. The conventional electrophoretic methods for phenotyping EAP can not compensate for diffusion, and the diffusion of each EAP band is not equal. Therefore, if one EAP band is more diffuse than another, mistyping could occur. [9] demonstrated that IEF of EAP in bloodstains is more sensitive than conventional typing methods. Due to the application of EAP phenotyping for forensics, paternity testing and other genetic studies, a new ultrathin-layer polyacrylamide gel isoelectric focusing (UL-PAGIF) method was developed that is reliable and more rapid and economical for EAP typing.Blood samples were obtained by finger prick from 8 1 selected individuals at the FBI Academy who were typed for EAP according to the method of Wraxall et al. [ 101. There were 10, 28,3 1,4,6 and 2 individuals expressing the A, B, BA, CA, CB and C phenotypes, respectively. Therefore, all common EAP phenotypes were tested for with this method. Bloodstains were prepared on washed cotton cloth, air dried and stored at -20 "C until analyzed. Cuttings (2.5 mm x 5.0 mm) of the bloodstains were extracted in 20 pl of 50 mM dithiothreitol for 30 min at room temperature. The extracts were absorbed onto 5 mm x 5 mm applicator tabs (LKB), blotted and applied 0.4 cm from the anode.Polyacrylamide gels (5 % T, 3 % C; 64 mm x 90 mm x 0.2 mm) were cast onto silanized glass plates using the flap technique [ 1 1, 121 and allowed to polymerize overnight. The gasket used for determining the 0.2 mm gel thickness was composed of 2 layers of Parafilm. The gradient was pH 5-7 (LKB) and the carrier ampholyte concentration was 4 % w/v. The anolyte and catholyte were 0.05 M H3P04 and 0.2 M NaOH. respectively. The distance between the electrode wicks was 5.4 cm. Gels were run either on the Ultrophor (LKB) at 4 "C or the Cold Focus Appartus (MRA). The UL-PAGIF conditions for gels on each IEF apparatus were outlined in Tables 1 and 2, respectively. After ULPAGIF, the

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Typing of esterase D by isoelectric focusing

Budowle

1984

Electrophoresis

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staining was negligible when using peroxidase-labelled probes, in contrast to the 1251-labelled antisera where the increased background staining reduced the sensitivity.The use of glutaraldehyde vapor fixation of the immobilized antigens followed by reduction with 0.02 % NaBH4 before performing the immunoassays (71 did not significantly improve the sensitivity, although slightly enhanced staining was observed.The conditions used in these immunoassays may differ from those used by other investigators and may be more or less sensitive compared to others. Therefore, the detection limits do not represent ultimate values, but serve as method to compare the various immunoassays. We conclude that the double sandwich immunoassay is the most attractive approach to detect antibody bound to nitrocellulose immobilized antigen since a) it is most sensitive technique and b), it reduces the number of incubation steps compared to other methods.

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Phosphoglucomutase‐1 subtyping of human bloodstains on ultrathin‐layer polyacrylamide gels

Cited by 26 publications

References 10 publications

Forensic science

Forensic science

Rapid electrofocusing of erythrocyte acid phosphatase

Typing of esterase D by isoelectric focusing

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