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

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Bu Makaleden Alıntı Yapın
Bimolecular Complementation Affinity Purification to Isolate Two Interacting Proteins. J. Vis. Exp. (Pending Publication), e21449, doi: (2023).

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