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Method Article

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

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July 8th, 2025

In This Article

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.

  1. 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 fluorescent protein (GFP) alone or GFP-fused Capn6 (calpain-6 gene) using cationic liposomes.
  2. Conjugate antibody to magnetic beads.
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Disclosures

No conflicts of interest declared.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
AcetonitrileWako014-00386
Citric acidWako030-05525
DTTNacalai tesque14112-94
Dynabeads protein GThermo Fisher Scientific10003D
Formic acidWako066-00461
IodoacetamideWako095-02151
Lipofectamine 3000Thermo Fisher ScientificL3000008
Living Colors A.v. Monoclonal Antibody (JL-8)Clontech632380
NaClWako191-01665
NH4HCO3Wako018-21742
Nonidet P-40SigmaN6507Poly(oxyethelene) octylphenyl ether (n=9)
Protease inhibitor cocktailSigmaP8465
Sequencing Grade Modified TrypsinPromegaV5111Trypsin
Sodium orthovanadateSigmaS6508
Sodium phosphate dibasic dihydrateSigma71643
TFAWako206-10731
TrisWako207-06275
Tween-20Wako160-21211

Tags

On Membrane DigestionProtein Protein InteractionCo ImmunoprecipitationPVDF MembraneTrypsin DigestionPeptide ExtractionMass SpectrometryNano LC HPLCDTT TreatmentIodoacetamide Alkylation