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Curr Protoc Mol Biol. 2001 May;Chapter 20:Unit 20.2. doi: 10.1002/0471142727.mb2002s33.

Affinity purification of proteins binding to GST fusion proteins.

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University of Texas Southwestern Medical Center, Dallas, Texas, USA.


This unit describes the use of proteins fused to glutathione-S-transferase (GST fusion proteins) to affinity purify other proteins, a technique also known as GST pulldown purification. The describes a strategy in which a GST fusion protein is bound to agarose affinity beads and the complex is then used to assay the binding of a specific test protein that has been labeled with [35S]methionine by in vitro translation. However, this method can be adapted for use with other types of fusion proteins; for example, His6, biotin tags, or maltose-binding protein fusions (MBP), and these may offer particular advantages. A describes preparation of an E. coli extract that is added to the reaction mixture with purified test protein to reduce nonspecific binding.

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