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



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Preparing Whole-Cell Lysates for Immunoblotting

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The purpose of preparing whole-cell lysates is to bring the proteins contained in the cells and tissue samples into a solution that can be loaded onto the gel for electrophoresis. The cells and tissues can be efficiently extracted by SDS-PAGE sample loading buffer or by other nondenaturing detergent-containing buffers. Here, the samples are prepared for electrophoresis using a standard Tris/glycine SDS-polyacrylamide gel (Laemmli and Quittner 1974). Extraction of cells using SDS-PAGE sample buffer is advantageous when rapid denaturation of the proteins in the sample is important, that is, when studying posttranslational modifications such as phosphorylation or ubiquitination that can be highly unstable in the extracts prepared under nondenaturing conditions.

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

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