On-Membrane Protein Digestion to Prepare Co-Immunoprecipitated Proteins for Interaction Studies

Published: May 31, 2023

Abstract

Source: Obama, T. et al., Evaluation of Protein–Protein Interactions using an On-Membrane Digestion Technique. J. Vis. Exp.  (2019)

This video demonstrates co-immunoprecipitated protein complexes on a PVDF membrane for protein-protein interaction analysis. The reduced proteins from complexes were treated with trypsin to cleave the individual proteins at the smaller peptides, then, the remaining peptides were extracted from the PVDF membrane. The pulled peptide derived from both proteins in the complex was then dried and resuspended in a low concentration of formic acid for further analysis.

Protocol

1. Immunoprecipitation NOTE: We used non-sodium dodecyl sulfate (SDS) lysis buffer and citrate elution, as described in the following sections. However, the use of an alternative in-house immunoprecipitation technique may be also applicable for preparing LC-MS/MS samples. Transfect cultured cells with vectors encoding an epitope tag alone or a fusion protein. For acquiring representative data, transfer J774 cells (1 x 106) with vectors encoding a green f…

開示

The authors have nothing to disclose.

Materials

Acetonitrile Wako 014-00386
Citric acid Wako 030-05525
DTT Nacalai tesque 14112-94
Dynabeads protein G Thermo Fisher Scientific 10003D
Formic acid Wako 066-00461
Iodoacetamide Wako 095-02151
Lipofectamine 3000 Thermo Fisher Scientific L3000008
Living Colors A.v. Monoclonal Antibody (JL-8) Clontech 632380
NaCl Wako 191-01665
NH4HCO3 Wako 018-21742
Nonidet P-40 Sigma N6507 Poly(oxyethelene) octylphenyl ether (n=9)
Protease inhibitor cocktail Sigma P8465
Sequencing Grade Modified Trypsin Promega V5111 Trypsin
Sodium orthovanadate Sigma S6508
Sodium phosphate dibasic dihydrate Sigma 71643
TFA Wako 206-10731
Tris Wako 207-06275
Tween-20 Wako 160-21211

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記事を引用
On-Membrane Protein Digestion to Prepare Co-Immunoprecipitated Proteins for Interaction Studies. J. Vis. Exp. (Pending Publication), e21397, doi: (2023).

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