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

Published: May 31, 2023

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. 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 li…

Açıklamalar

The authors have nothing to disclose.

Materials

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

Etiketler

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Bu Makaleden Alıntı Yapın
ELISA-Based Method to Analyze Interactions between Bait and Prey Proteins. J. Vis. Exp. (Pending Publication), e21401, doi: (2023).

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