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

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…

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