Optimization of Formaldehyde Cross-Linking for Protein Interaction Analysis of Non-Tagged Integrinβ1

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 β1 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 β1 was found in a high molecular weight complex after formaldehyde cross-linking. Eight different anti-integrin β1 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 β1, α4 and α6 or β1, α6, α2, and α5, respectively.

DIRAS3 regulates the autophagosome initiation complex in dormant ovarian cancer cells

“…56 Cells were incubated with formaldehyde solution with mild agitation for 10 min and quenched with ice-cold 1.25 M glycine in PBS, and then cells were washed with glycine in PBS and solubilized with lysis buffer. Co-immunoprecipitation and western blotting were performed as describe in the figure legends.…”

Direct Interaction of Baculovirus Capsid Proteins VP39 and EXON0 with Kinesin-1 in Insect Cells Determined by Fluorescence Resonance Energy Transfer-Fluorescence Lifetime Imaging Microscopy

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Regulator of G Protein Signaling 7 (RGS7) Can Exist in a Homo-oligomeric Form That Is Regulated by Gαo and R7-binding Protein

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