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Distler JJ, Patel R, Jourdian GW. Immobilization and assay of low-molecular-weight phosphomannosyl receptor in multiwell plates. Anal Biochem. 1987;166(1):65-71doi: 10.1016/0003-2697(87)90546-x.
Distler, J. J., Patel, R., & Jourdian, G. W. (1987). Immobilization and assay of low-molecular-weight phosphomannosyl receptor in multiwell plates. Analytical biochemistry, 166(1), 65-71. https://doi.org/10.1016/0003-2697(87)90546-x
Distler, J J, et al. "Immobilization and assay of low-molecular-weight phosphomannosyl receptor in multiwell plates." Analytical biochemistry vol. 166,1 (1987): 65-71. doi: https://doi.org/10.1016/0003-2697(87)90546-x
Distler JJ, Patel R, Jourdian GW. Immobilization and assay of low-molecular-weight phosphomannosyl receptor in multiwell plates. Anal Biochem. 1987 Oct;166(1):65-71. doi: 10.1016/0003-2697(87)90546-x. PMID: 2823641.
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Anal Biochem. 1987 Oct;166(1):65-71. doi: 10.1016/0003-2697(87)90546-x.

Immobilization and assay of low-molecular-weight phosphomannosyl receptor in multiwell plates.

Analytical biochemistry

J J Distler, R Patel, G W Jourdian

Affiliations

  1. Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor 48109.

PMID: 2823641 DOI: 10.1016/0003-2697(87)90546-x

Abstract

A novel sensitive binding assay for quantitation of a low-molecular-weight phosphomannosyl receptor (41-46 K) was devised. The receptor was immobilized by immunochemical means in the wells of polystyrene multiwell plates. The lysosomal enzyme ligand, testicular beta-galactosidase, was added and receptor-bound beta-galactosidase was measured by conventional colorimetric analysis using p-nitrophenyl beta-galactoside as substrate. Inhibitors of the binding of beta-galactosidase to the receptor were removed prior to addition of beta-galactosidase and did not interfere with the assay. Low-molecular-weight phosphomannosyl receptor was readily quantitated in the range of 4 to 100 ng of receptor protein. Binding of beta-galactosidase to the receptor was specifically inhibited by 5 mM mannose 6-phosphate. The receptor exhibited optimum binding of beta-galactosidase at pH 5.7 and was saturated with beta-galactosidase at 320 munits/ml in the presence of 20 mM MnCl2. The requirement for MnCl2 was greatly diminished at higher concentrations of beta-galactosidase. Application of the assay procedure to the quantitation of the low-molecular-weight phosphomannosyl receptor in mammalian tissues is discussed.

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