Human VE Cadherin ELISA Kit (ab210968)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-Equilibrate all reagents to room temperature (18-25°C) prior to use.	-Avoid foaming or bubbles when mixing or reconstituting components. -Ensure plates are properly sealed or covered during incubation steps. -Complete removal of all solutions and buffers during wash steps is necessary to minimize background.	-Unused well strips should be returned to the plate packet and stored at 4°C.

Upstream tips
-Equilibrate all reagents to room temperature (18-25°C) prior to use.
Protocol tips
-Avoid foaming or bubbles when mixing or reconstituting components. -Ensure plates are properly sealed or covered during incubation steps. -Complete removal of all solutions and buffers during wash steps is necessary to minimize background.
Downstream tips
-Unused well strips should be returned to the plate packet and stored at 4°C.

Publication protocol

To verify the results of protein microarray analysis, the most differentially expressed proteins (> fivefold) were validated by ELISA. A high-binding 96-well plate was pre-coated with 100 µl of appropriate antibodies (1 µg/ml diluted in carbonate buffer) at 4 °C overnight. The following antibodies were used in this step: CXC chemokine ligand 16 (CXCL16, Invitrogen, cat #MA5-23869), IL-2Rα (Abcam, cat #ab46036), IL-2Rγ (R&D Systems, cat #YD1104), MMP-1 (Abcam, cat #ab100603), MMP-9 (Abcam, cat # ab100610), PDGFR-α (Abcam, cat #ab65258), SDF-1a (Abcam, cat #ab100637), TGF-β (Abcam, cat #ab100647), platelet endothelial cell adhesion molecule (PECAM)-1 (Abcam, cat #ab190814), and vascular endothelial (VE)-cadherin (Abcam, cat #ab210968). After washing with PBST twice, the plate was blocked by blocking solution for 1 h at room temperature and then incubated with 100 µl of the cell medium (in duplicate) used for protein microarray for 1 h at 37 °C. The plate was washed with PBST and incubated with diluted detection antibody for 1 h at room temperature, followed by an incubation with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at 37 °C. After thorough washing with PBST, the plate was incubated with 100 µl of substrate TMB solution for 15–30 min at 37 °C in darkness. Then 50 µl of stop solution was added to terminate color development. The plate was read with a microplate reader at the absorbance of 450 nm. The OD value was calculated using blank control space as zero reference. At the same time, the standard curve was drawn according to the results for the positive control with known concentrations, and then the concentration of each protein was calculated according to the correlation coefficient and the formula of the standard curve.

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Manufacturer protocol

Download the product protocol from Abcam for Human VE Cadherin ELISA Kit (ab210968) below.

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