ELISA-Based Method to Analyze Interactions between Bait and Prey Proteins

Abstract

Source: Singh, B. et al., Assays for Studying the Role of Vitronectin in Bacterial Adhesion and Serum Resistance. J. Vis. Exp. (2018)

This video describes an ELISA-based technique that detects protein-protein interactions between two proteins by measuring the binding of the second protein to the immobilized first protein. The bound second protein is detected using specific antibodies labeled with an enzyme, such as horseradish peroxidase.

Protocol

1. ELISA analysis of the interaction between recombinant PH and Vn

NOTE: Controls need to be included to exclude nonspecific binding. Human Factor H (FH) or C4b-binding protein (C4BP) are used as positive and negative controls, respectively.

1. Dilute each of the human proteins (Vn, FH, and C4BP) separately to 50 nM in Tris-HCl, pH 9.0 (coating buffer). Dispense 100 µL of protein solution into each well of a Polysorp microtiter plate. Close the plates with the lid and store them at 4 °C overnight (16 h) to facilitate the immobilization of protein onto microtiter plate wells.
2. Discard the solution....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
AR2G sensors	Pall Life Science	18-5095	Sensor to immobilized protein by amino coupling
Acetone	VWR	97064-786	Analysis grade
Bovine Serum Albumins (BSA)	Sigma-Aldrich	A2058	Suitable for cell culture
C4BP (C4b-binding protein)	Complement Technology, Inc.	A109	Bought as Frozen liquid form
Citric acid	Sigma-Aldrich	251275-500G	American Chemical Society (ACS) grade
HRP-conjugated anti-His tag antibodies	Abcam	ab1269	Polyclonal
C4BP	Complement Technology, Inc.	A109	Frozen solution
Histidine affinity resin column (HisTrap HP)	GE Health Care Life Science	17-5247-01	Columns prepacked with Ni Sepharose
His-tagged PH			Recombinantly expressed and purified in our lab
Methano	VWR	BDH1135-1LP	Analysis grade
Plasmid containing C-terminal 6x His-tag on the backbone (pET26(b))	Novagen	69862-3	DNA vector
Polysorb microtitre plates	Sigma-Aldrich	M9410	For ELISA
Tetramethylbenzidine	Sigma-Aldrich	860336-100MG	ELISA grade
Vitronectin (Vn) from human plasma	Sigma-Aldrich	V8379-50UG	Cell culture grade