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

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Preparing Protein Solutions for Immunoblotting

(Protocol summary only for purposes of this preview site)

Most protein solutions can be mixed with sample buffer and analyzed directly by immunoblotting. Any protein extraction buffer or solvent that is compatible with gel electrophoresis can be used for preparing samples for immunoblotting. Examples of the protein solutions suitable for immunoblotting include purified proteins, detergent lysates of cells, samples prepared by subcellular fractionation, and protein eluates from chromatography columns. The protein of interest must be sufficiently abundant in the sample to allow for efficient detection (see Detection Methods in the chapter Introduction), but the total amount of protein in the sample should not exceed the loading capacity of the gel (see Protocol 4). For this reason, protein antigens of very low abundance must be partially purified from the protein mixtures containing high concentrations of the contaminating proteins. In this protocol, solutions containing the protein antigens of interest are mixed with SDS-PAGE sample buffer and boiled to prepare samples for protein gel electrophoresis.

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

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