Far western blot

A Far Western blot is a biochemical method used to detect protein-protein interactions .

principle

A sample is separated by SDS-PAGE or 2D gel electrophoresis and transferred to a membrane by Western blot . The blot is after blocking with a purified protein incubated. A specific antibody for immunostaining is required for detection of this protein , or the protein has previously been labeled with a radionuclide , by biotinylation or with a fluorophore . By detecting the protein that has bound to its interaction partner on the blot, the molar masses of the binding partners are obtained by comparison with the size marker and also an identifiable partial sequence by mass spectrometry or Edman degradation .

Since some protein folds are not restored due to the denaturation in SDS-PAGE and the subsequent, often incomplete renaturation , this method is suitable for interactions that are not sensitive to denaturation, such as e.g. B. linear or phosphorylated sequences. Alternatively, a native gel electrophoresis can be carried out to separate the proteins while maintaining the native protein structure , but size determination using the size marker is not possible.

literature

Erica A. Golemis, Peter D. Adams: Protein-protein interactions. A molecular cloning manual. 2nd edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2005, ISBN 0-87969-723-7 .
Friedrich Lottspeich , Joachim W. Engels (Ed.): Bioanalytik. 3rd, (updated and expanded) edition. Springer Spectrum, Berlin a. a. 2012, ISBN 978-3-8274-2942-1 .

Individual evidence

↑ Catherine S. Chan, Tara ML Winstone, Raymond J. Turner: Investigating protein-protein interactions by far-Westerns. In: Advances in Biochemical Engineering / Biotechnology. Vol. 110, 2008, ISSN 0724-6145 , pp. 195-214, PMID 18219468 , doi : 10.1007 / 10_2007_090 .
↑ Randy A. Hall: Studying protein-protein interactions via blot overlay or Far Western blot. In: Methods in Molecular Biology. Vol. 261, 2004, ISSN 1064-3745 , pp. 167-174, PMID 15064457 , doi : 10.1385 / 1-59259-762-9: 167 .

[1] Catherine S. Chan, Tara ML Winstone, Raymond J. Turner: Investigating protein-protein interactions by far-Westerns. In: Advances in Biochemical Engineering / Biotechnology. Vol. 110, 2008, ISSN 0724-6145 , pp. 195-214, PMID 18219468 , doi : 10.1007 / 10_2007_090 .

[2] Randy A. Hall: Studying protein-protein interactions via blot overlay or Far Western blot. In: Methods in Molecular Biology. Vol. 261, 2004, ISSN 1064-3745 , pp. 167-174, PMID 15064457 , doi : 10.1385 / 1-59259-762-9: 167 .