Bimolecular Complementation Affinity Purification to Isolate Two Interacting Proteins
Published: June 29, 2023
Abstract
Source: Hastings, J. F. et al. Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP). J. Vis. Exp. (2018)
In this video, we have demonstrated bimolecular complementation affinity purification, BiCAP, to detect and isolate specific protein dimers using conformation-specific single-domain antibodies.
Protocol
1. Cell Culture and Transfection NOTE: For transfection of the BiFC vectors it is important to achieve a high efficiency and relatively homogenous transfection. The vectors will likely be compatible with any standard transfection reagent, and the transfection conditions should be optimized accordingly. To perform mass spectrometry, we usually culture cells within 10 cm dishes, although this can also be proportionally scaled down to smaller dishes or plates for e…
Açıklamalar
The authors have nothing to disclose.
Materials
| DMEM | Gibco | 11995-073 | Cell culture |
| FBS | Life Technologies Australia | 10099-141 | Cell culture |
| Penicillin/Streptomycin | Life Technologies Australia | 15070-063 | Cell culture |
| jetPRIME transfection buffer | Polyplus | 114-15 | Cell culture |
| jetPRIME transfection reagent | Polyplus | 114-15 | Cell culture |
| PhosSTOP (Phosphatase inhibitor) | Sigma-Aldrich | 4906837001 | Cell culture |
| Complete, Mini, EDTA-free Protease inhibitor cocktail | Roche | 11873580001 | Plastic ware |
| Cell Scraper | Sarstedt | 83.1832 | |
| GFP-Trap_A | Chromotek Gmbh | gta-100 | GFP nanobody coupled to agarose beads |
| N-terminal GFP monoclonal antibody | Covance | MMS-118P | Will detect the V1 tag within the BiFC vectors |
| C-terminal GFP monoclonal antibody | Roche | 11814460001 | Will detect the V2 tag within the BiFC vectors |
| Sample buffer | Invitrogen | NP0008 | Supplemented with 1 mL β-mercaptoethanol. |
| Sequencing-grade modified trypsin | Promega Corporation | V5117h | Cell culture |
| LoBind microcentrifuge tubes | Point of Care Diagnostics | 0030 108 116 | Plastic ware |
| Iodoacetamide | Sigma-Aldrich | I1149-5G | Prepared at 5 mg/mL in ultrapure water |
| LTQ Orbitrap Velos Pro | Thermo Fisher | ||
| DH5α cells | Thermo Fisher | Heat-shock-competent cells |
Etiketler
Bu Makaleden Alıntı Yapın
Bimolecular Complementation Affinity Purification to Isolate Two Interacting Proteins. J. Vis. Exp. (Pending Publication), e21449, doi: (2023).