Glycoprotein Methods and Protocols - P3

Detection and Quantitation of Mucins 45

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From: Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The Mucins

Edited by: A. Corfield © Humana Press Inc., Totowa, NJ

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Detection and Quantitation of Mucins

Using Chemical, Lectin, and Antibody Methods

Michael A. McGuckin and David J. Thornton

1. Introduction

Detection and quantitation of mucins can be important in both the research and

clinical settings. Applications may range from detection of potentially novel mucins

present during purification from mucus, to quantitation of specific mucin core pro￾teins or carbohydrate moieties present in clinical samples. This chapter discusses pro￾cedures and limitations of several different strategies available to detect and quantify

these glycoproteins from biological samples, with a view to providing guidelines from

which to select the best applicable techniques. Example protocols are then provided to

give a starting point for development of a technique. Refer to Chapter 3 for detection

of mucins in histological preparations (1); note, however, that many of the principles

for selection of detection tools discussed herein are applicable to histological detection.

Because of the extreme size and extent of glycosylation of mucins, coupled with

the fact that many secreted mucins are capable of forming gels, these glycoproteins

can be quite difficult to work with biochemically. It is therefore extremely important

before attempting to detect mucins that the researcher has a good understanding of the

behavior of these molecules in solution, particularly with regard to their potential lack

of solubility in standard physiological buffers. Because of these properties, standard

preparative methods for secreted mucins involve extraction in chaotropic agents (usu￾ally 6 M guanidinium chloride) and purification in CsCl density gradients in either the

presence or absence of 4 M guanidinium chloride. Therefore, methods often have to be

applicable to assay in the presence of high concentrations of these agents. Failure to

adhere to these considerations may result in embarrassing false-negative results. Read￾ers are advised to refer to Chapters 1 and 2 (2,3) of this volume for the preparation of

secreted and membrane-associated mucins, respectively, and to Chapter 7 (4) for a

discussion of methods for mucin separation.