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…
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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 |
