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