James D San Antonio and Arthur D Lander 1 Introduction

Affinity coelectrophoresis (ACE) was developed as a tool to measure the strengths of interaction between proteoglycans (PGs) or glycosaminoglycans (GAGs) and proteins, and to assess the specificity of the interaction (i.e., to detect and fractionate GAG or PG sample constituents that differentially bind to protein) (1). In ACE, trace concentrations of radiolabeled GAG or PG are subjected to electrophoresis through agarose lanes containing protein at various concentrations. The electrophoretic pattern of the radiolabeled GAG or PG is then visualized by autoradiography, or using a phosphorimager, and the apparent dissociation constant (Kd) is calculated as the protein concentration at which the GAG or PG is half-shifted from being fully mobile at very low protein concentrations (or between protein-containing lanes) to being maximally retarded at saturating protein concentrations (see Figs. 1-3).

ACE holds many advantages over other means of studying GAG or PG-protein interactions since it: (1) uses only trace quantities of the interacting molecules (typically a microgram or less of GAGs, and a milligram or less of protein); (2) studies behaviors of native proteins and of radiolabeled PGs or GAGs that can be essentially unmodified (e.g., through metabolic radiolabeling) or minimally modified (e.g., by radioiodination); (3) can measure strengths of binding even for relatively weak interactions characteristic of GAG or PG-protein interactions (e.g., 100 nM or weaker Kd); (4) can detect protein-binding heterogeneity in a GAG or PG population and can even be used to isolate the differentially binding subpopulations for further analysis (see Fig. 4); and (5) is a low cost, simple, and rapid method, and is amenable to quantitative analysis.

The theory of ACE has been described in detail elsewhere (1,2) and will not be repeated here. Rather, this chapter will serve as a description of ACE methods, and will also include a protocol for the preparation of radioiodinated heparin samples, which are often useful in various ACE applications. However, before attempting ACE, there are several important questions which need to be addressed:

From: Methods in Molecular Biology, Vol. 171: Proteoglycan Protocols

Edited by: R. V. Iozzo © Humana Press Inc., Totowa, NJ

PG or GAG i

High [protein] _Low [protein]

Before

High [protein] _Low [protein]

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