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Methods Mol Biol. 2019;1855:417-436. doi: 10.1007/978-1-4939-8793-1_35.

SDS-PAGE for 35S Immunoprecipitation and Immunoprecipitation Western Blotting.

Author information

1
Arthritis and Clinical Immunology, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA.
2
Arthritis and Clinical Immunology, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA. Ira-Targoff@omrf.org.
3
Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA. Ira-Targoff@omrf.org.
4
Veterans Affairs Medical Center of Oklahoma City, Oklahoma City, OK, USA. Ira-Targoff@omrf.org.

Abstract

This report discusses recent methods of sample preparation and gel electrophoresis for 35S immunoprecipitation (IP) and IP western blotting. In both methods, IP is used to obtain purified proteins, and sodium dodecyl sulfate polyacrylamide gel electrophoresis is used to separate the proteins on a gel. In 35S IP, the proteins are radiolabeled and visualized on film by fluorography; in IP blotting, proteins are transferred onto nitrocellulose paper, and antibodies are used to detect specific proteins. A similar IP and SDS-PAGE method can be used for both procedures, but IP blotting has the potential advantages of improvement in sensitivity for low-abundance proteins and enhanced specificity for identification of proteins from a mixture. Some of the technical adaptations discussed here to facilitate IP blotting and avoid loss of beads or purified proteins may also be useful for 35S IP.

KEYWORDS:

35S immunoprecipitation; Autoantibody; Autoantigen; Dimethyl pimelimidate; Immunoblot (Western blot); Immunoprecipitation; Isotachophoresis; SDS-PAGE

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