PD-1:PD-L2[Biotinylated] Inhibitor Screening Colorimetric Assay Kit
The PD1: PD-L2[Biotinylated] Inhibitor Screening Colorimetric Assay Kit is a colorimetric assay designed for screening and profiling molecules that block the binding of PD-1 (programmed cell death protein 1) and PD-L2 (programmed cell death 1 ligand 2). This assay kit comes in a convenient 96-well format, with enough purified PD-1 (amino acids 25-167), biotin-labeled PD-L2 (amino acids 20-219), assay buffers and detection reagents for 100 enzyme reactions.
The key to this kit is the high affinity of biotin-labeled PD-L2 for streptavidin-HRP. First, a 96-well plate is coated with PD-1, followed by incubation with PD-L2. Finally, the plate is treated with streptavidin-HRP followed by addition of a colorimetric HRP substrate to produce color, which can then be measured using a UV/Vis spectrophotometer microplate reader. The signal generated is proportional to the binding between PD-1 and PD-L2.
- 1x PBS (Phosphate Buffer Saline) Buffer
- UV/Vis spectrophotometer microplate reader capable of reading absorbance at λ=450 nm*
- 1 N hydrochloric acid (aqueous)
- Adjustable micropipettor and sterile tips
- Orbital Shaker
*Alternately, a spectrophotometer reading at 650 nm may be used, but the sensitivity of the assay will be greatly reduced
| Catalog # | Name | Amount | Storage |
| 71106 | PD-1 (CD279), Fc fusion (Human)* | 10 µg | -80°C |
| 71108 | PD-L2 (CD273), Fc fusion, Biotin-labeled (Human)* | 5 µg | -80°C |
| 79311 | 3x Immuno Buffer 1 | 50 ml | -20°C |
| 79728 | Blocking Buffer 2 | 50 ml | +4°C |
| 79742 | Streptavidin-HRP | 10 µl | +4°C |
| 79651 | Colorimetric HRP Substrate | 10 µl | +4°C |
| 79964 | 96-well transparent plate | 1 | Room Temp |
*The concentration of the protein is lot-specific and will be indicated on the tube.
PD-L1 and PD-L2 binding to PD-1, a receptor expressed on T-cells, negatively regulates immune responses. PD-1 ligands PD-L1 and PD-L2 are found on the surface of many cancer cells, and their interaction with receptor PD-1 inhibits T cell activity and allows cancer cells to escape immune surveillance. This pathway is also involved in regulating autoimmune responses. Therefore, these proteins (termed immune checkpoints) are promising therapeutic targets for many types of cancer as well as multiple sclerosis, arthritis, lupus, and type I diabetes. Checkpoint inhibitors have remarkable efficacy in a wide range of cancer types and have revolutionized cancer treatment. PD-1 inhibitors nivolumab, pembrolizumab, cemiplimab and PD-L1 inhibitors atezolizumab, avelumab, and durvalumab are all FDA-approved drugs for immuno-therapy.
Sasca D, et al. 2019 Blood 133: 2305-2319.
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