| Assay Method Information | |
| | VEGFR2 |
| Description: | Adult human umbilical vein endothelial cells (HUVEC) (Cat# CC-2519, Lonza) were seeded into clear-bottom, TC treated 12 well plates (Cat #665180, Greiner Bio-One) in EGM-2 (Cat# CC-3162, Lonza) at 180,000 cells/well (volume 1 mL), and the plates incubated at 37° C. and 5% CO2 for 6 hours. The media was replaced with EBM-2 (Cat # CC-3156, Lonza)+0.1% BSA (Cat# A8412, Sigma) and cells incubated for a further period (overnight at 37° C. and 5% CO2).96 well Maxisorp immuno plates (Cat #439454, Nunc) were coated with 100 L of Total VEGFR2 capture antibody (Part #841434, Human Total VEGFR2/FLT4 ELISA Kit, Cat # DYC1780, R&D Systems), or Phospho VEGFR2 Capture antibody (Part #841419, Human Phospho VEGFR2/FLT4 ELISA Kit, Cat# DYC1766, R&D Systems). The plates were covered and incubated at room temperature overnight. The coating antibody was flicked out and the plates washed three times with Wash Buffer (Phosphate buffered saline (137 mM NaCl, 2.7 nM KCl, 8.1 nM Na2HPO4, 1.5 mL KH2PO4, pH 7.2-7.4), 0.05% Tween 20). 300 uL of blocking buffer (5% v/v Tween 20, 5% w/v sucrose in PBS) was then added to wells and plate incubated for 2 hours at room temperature. |
| Affinity data for this assay | |
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