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…
Divulgaciones
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))