* Suggested working dilutions are given as a guide only. It is recommended that the user titrates the product for use in their own experiment using appropriate negative and positive controls.
Product Specific Information
The VEGF-A monoclonal antibody clone # 16F1 (Product # M808) has successfully been paired as the coating antibody in a sandwich ELISA with detection antibody P802B (biotinylated conjugate of Product # P802).
Typical dilutions for sandwich ELISA include 1µg/ml for coating and 0.125-0.5µg/ml for detection.
The VEGF gene is a member of the PDGF/VEGF growth factor family and encodes a protein that is often found as a disulfide linked homodimer. This protein is a glycosylated mitogen that specifically acts on endothelial cells and has various effects, including mediating increased vascular permeability, inducing angiogenesis, vasculogenesis and endothelial cell growth, promoting cell migration, and inhibiting apoptosis.
VEGF-A Antibody (M808) in ELISA
ELISA with anti-VEGF-A Monoclonal Antibody [16F1] (M808)
Sandwich ELISA analysis of Human VEGF was performed using the Thermo Scientific ELISA Kit (Product # EHVEGF) by coating a blank 96-well microtiter plate with 100µl per well of Human VEGF moncolonal antibody (Product # M808) in duplicate at 1, 3, 4, 5, 7, and 9µg/ml in DPBS (Product # 28374) and incubating for 12-18 hours at 4ºC. The plate was aspirated and blocked with 300µl per well of 4% BSA and 5% sucrose in DPBS for 1 hour at room temperature. Human VEGF recombinant protein (Product # SVEGF) at 50µl per well (in addition to 50µl of sample diluent per well) was added in duplicate at 2000, 1000, 500, 250, 125, 62.5, 31.25, and 0pg/ml for 2 hours at room temperature. The plate was washed with ELISA Wash Buffer (Product # N503) and incubated with 100µl of Human VEGF biotinylated polyclonal antibody (Product # P802B) in all applicable wells at 0.5µg/ml for 1 hour at room temperature. The plate was washed and incubated with 100µl per well of Streptavidin-HRP (Product # N504) in all test wells at 1:40,000 dilution for 30 minutes at room temperature and then washed and incubated with 100µl per well of TMB substrate (Product # 34028) for 30 minutes at room temperature in the dark. The plate was stopped with 0.16M sulfuric acid (Product # N600). Absorbances were read on a spectrophotometer at 450-550nm.
VEGF-A Antibody (M808) in WB
Western Blot with anti-VEGF-A Monoclonal Antibody [16F1] (M808)
Western blot analysis of Human VEGF was performed by loading 0. 5µg of VEGF transfected 293T cell culture supernatant and recombinant human VEGF onto a 4-12% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked with 3% BSA/TBST for at least 1 hour. The membrane was probed with a human VEGF monoclonal antibody (Product # M808) at a dilution of 1µg/ml overnight at 4°C on a rocking platform, washed in TBS-0.1%Tween 20 and probed with a goat anti- mouse-HRP secondary antibody (Product # 31430) at a dilution of 1:15,000 for at least one hour. Chemiluminescent detection was performed using SuperSignal West Dura (Product #34075).
(This product is for In Vitro experimental use only. Not for resale without express authorization.)