TY - JOUR
T1 - Affinity purification of proteins binding to GST fusion proteins.
AU - Swaffield, J. C.
AU - Johnston, S. A.
PY - 2001/5
Y1 - 2001/5
N2 - 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.
AB - 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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U2 - 10.1002/0471142727.mb2002s33
DO - 10.1002/0471142727.mb2002s33
M3 - Article
C2 - 18265191
AN - SCOPUS:48849100969
SN - 1934-3639
VL - Chapter 20
SP - Unit 20.2
JO - Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.]
JF - Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.]
ER -