Optimization of formaldehyde cross-linking for protein interaction analysis of non-tagged integrin beta1.

Journal of Biomedicine & Biotechnology
Cordula Klockenbusch, Juergen Kast

Abstract

Formaldehyde cross-linking of protein complexes combined with immunoprecipitation and mass spectrometry analysis is a promising technique for analysing protein-protein interactions, including those of transient nature. Here we used integrin beta1 as a model to describe the application of formaldehyde cross-linking in detail, particularly focusing on the optimal parameters for cross-linking, the detection of formaldehyde cross-linked complexes, the utility of antibodies, and the identification of binding partners. Integrin beta1 was found in a high molecular weight complex after formaldehyde cross-linking. Eight different anti-integrin beta1 antibodies were used for pull-down experiments and no loss in precipitation efficiency after cross-linking was observed. However, two of the antibodies could not precipitate the complex, probably due to hidden epitopes. Formaldehyde cross-linked complexes, precipitated from Jurkat cells or human platelets and analyzed by mass spectrometry, were found to be composed of integrin beta1, alpha4 and alpha6 or beta1, alpha6, alpha2, and alpha5, respectively.

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Citations

May 23, 2012·Analytical and Bioanalytical Chemistry·Cordula KlockenbuschJuergen Kast
Dec 18, 2013·The Journal of Cell Biology·Nina SchweizerHelder Maiato
Dec 15, 2015·The Journal of Physical Chemistry. B·Flaviyan Jerome IrudayanathanShikha Nangia
Apr 23, 2016·Scientific Reports·Christian FercherWalter Keller
Jan 10, 2017·Journal of Cellular Biochemistry·Sung-Jin BaeKyu-Won Kim
Jan 20, 2017·Science Translational Medicine·Raquel Lima E SilvaPeter A Campochiaro
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Methods Mentioned

BETA
immunoprecipitation
pull-down
GTPase
immunoprecipitations
electrophoresis
pull-downs
affinity purification
pull down
coimmunoprecipitation

Software Mentioned

ImageJ
SWISS MODEL Viewer
Mascot
ENSEMBL

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