Antibodies, A Laboratory Manual, Second Edition, Edited by Edward A. Greenfield



Table of Contents

Expand All | Contract All

Labeling Antibodies Using a Maleimido Dye

(Protocol summary only for purposes of this preview site)

Fluorophore-maleimide derivatives are effective for labeling sulfhydryl-containing molecules. Maleimide groups react with free thiols at pH 6.57.5 forming a covalent bond (see the subsection Sulfhydrals in the Introduction). Reducing agents should be avoided during the conjugation step. This protocol uses the cross-linker N-succinimidyl S-acetylthioacetate (SATA) to introduce thiol groups on the antibody while maintaining the divalent nature of the antibody. Alternatively, the antibody can be digested and reduced to monovalent Fab fragments, which can then be labeled directly with maleimido derivatives (see Protocol 3). Typically, a 1015 molar excess of the fluorophore derivative is used. Actual amounts will depend on the molecular weight of the dye.

Antibodies: A Laboratory Manual, Second edition
Antibodies: A Laboratory Manual, Second edition
Antibodies: A Laboratory Manual, Second edition

Search for information about other protocols included in the book: