Anti-VEGFA antibody (ab46154)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-These buffers may be stored at 4°C for several weeks or aliquoted and stored at -20°C for up to a year.	-Rabbit (1:1000) -To reduce and denature your samples, boil each cell lysate in sample buffer at 100°C for 5 min. Lysates can be aliquoted and stored at -20°C for future use.

Upstream tips
-These buffers may be stored at 4°C for several weeks or aliquoted and stored at -20°C for up to a year.
Protocol tips
-Rabbit (1:1000) -To reduce and denature your samples, boil each cell lysate in sample buffer at 100°C for 5 min. Lysates can be aliquoted and stored at -20°C for future use.

Publication protocol

After treatment with FF with or without S1P, EA.hy926 endothelial cells were lysed in lysis buffer (20 mM Tris–HCl pH 8, 137 mM NaCl, 1% Nonidet P-40 and 10% glycerol) supplemented with protease inhibitors (0.5 mM PMSF, 0.025 mMN-CBZ-l-phenylalanine chloromethyl ketone, 0.025 mMN-p-tosyl-lysine chloromethyl ketone and 0.025 mM l-1-tosylamide-2-phenyl–ethylchloromethyl ketone). The cell lysates were centrifuged at 10 000g for 10 min at 4°C. Rat ovaries were resuspended in five volumes of buffer lysis and homogenized with an Ultra-Turrax (IKA Werk, Breisgau) homogenizer. Ovarian samples were then centrifuged at 4°C for 10 min at 10 000g and the resulting pellets were discarded. Protein concentration was measured by the Bradford assay. After boiling for 5 min, 40 μg of protein was applied to a 12% SDS–polyacrylamide gel, and electrophoresis was performed at 25 mA for 1.5 h. The resolved proteins were transferred for 2 h onto nitrocellulose membranes. The blot was preincubated in blocking buffer (5% nonfat milk, 0.05% Tween 20 in 20 mM TBS pH 8.0) for 1 h at room temperature and incubated overnight in blocking buffer at 4°C with appropriate primary antibodies: β-actin 1/3000 (sc-1616-R), N-cadherin 1/200 (sc-7939), vascular endothelial (VE)-cadherin 1/100 (sc-9989) and β-catenin 1/200 (sc-59737) (Santa Cruz Biotechnology, Inc., Santa Cruz, USA), phospho-VE-cadherin 1/1000 (MAB1002) (Millipore, MA, USA), VEGF 1/1000 (ab46154) (Abcam, Cambridge, USA), KDR (VEGFR-2) 1/1000 (D5B1) (Cell Signaling Technology, Inc., Danvers, MA, USA). The protein levels were analyzed by densitometry using Scion Image for Windows (Scion Corporation, Worman's Mill, CT, USA). Optical density data are expressed as arbitrary units ± SEM.

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Manufacturer protocol

Download the product protocol from Abcam for Anti-VEGFA antibody (ab46154) below.

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