Human angiogenesis antibody array kit

The effect of the n-butanol fraction on the expression levels of angiogenesis and metastasis-related proteins was assayed using the human angiogenesis antibody array kit (R&D Systems, USA), as described by the manufacturer (R&D Systems, Minneapolis, MN, USA). Briefly, 300 µg of protein from each sample was incubated with the angiogenesis array membranes at 4°C overnight. The membranes were then washed with an array wash buffer for 5 minutes at room temperature. The Streptavidin-HRP solution was added to the membranes which were then incubated for 30 minutes at room temperature on an orbital shaker. Pictures of the arrays were captured using the ChemiDoc XRS and the signal density of each spot representing the protein of interest was determined using imager Quantity One software (Bio-Rad Laboratories, USA). The background signal was subtracted from the total signal value of each spot and each pair of duplicate spots was averaged for each protein in each treatment group.

Passage 4–6 cells were cultured for 9 days in growth factor deprivation medium; 1.5 ml of supernatant from the clinical case 3 C-FVM was collected, and protein secretion was analyzed using the Human Angiogenesis Antibody Array Kit (R&D Systems, Minneapolis, MN). The membrane, containing antibodies for 55 proteins related to angiogenesis, was immersed in 1.5 ml of 2:1 supernatant and array buffer (included in the kit), as well as 15 µl of reconstituted antibody cocktail. Following incubation in blocking buffer for 1 h, the membrane was incubated overnight on a rocking platform at 4 °C, washed three times for 10 min, submerged in 2 ml of diluted streptavidin-horseradish peroxidase (HRP) in array buffer, and incubated for 30 min at room temperature. Following a rinse in the wash buffer, the membrane was coated with 1 ml of the chemireagent mix. Kodak BioMax light film (Cat. No. 1788207, Eastman Kodak Company, Rochester, NY) was placed over the membrane and allowed to sit in the dark for 30 min. The film was then processed and developed using a Kodak M35A film processor (Eastman Kodak Company) and scanned with an Epson Perfection V500 (Long Beach, CA) photo scanner.