Bimolecular Complementation Affinity Purification to Isolate Two Interacting Proteins

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 experiments that require less material.

1. Seed 1.0 × 10⁶ HEK293T cells in a 10 cm dish with 10 mL of DMEM media, supplement....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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