Human vegf quantiglo elisa kit

After 48 h of transfection, conditioned medium was removed and analyzed by ELISA for detection of secreted VEGF-A (human VEGF QuantiGlo ELISA kit, R&D Systems, Minneapolis, MN, USA). In parallel, whole-cell lysates were prepared with radioimmunoprecipitation assay lysis buffer supplemented with protein enzyme inhibitors. The protein concentration was determined by use of the bicinchoninic acid assay. Equal amounts of protein (20 ug per lane) were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to poly(vinylidene difluoride) membranes. The membranes were blocked with 5% fat-free milk in tris(hydroxymethyl)aminomethane-buffered saline (TBS) (pH 7.5) for 1 h at room temperature, and then incubated with anti-VEGF-A (ab9570, Abcam), anti-entothelin 1 (ab113697, Abcam), anti-MET (sc-8307, Santa Cruz Biotechnology), or anti-GAPDH (sc-47724, Santa Cruz Biotechnology) antibodies overnight at 4 °C. After incubation with the appropriate horseradish peroxidase conjugated secondary antibody (Pierce) for 1 h at room temperature, membranes were incubated with enhanced chemiluminescence reagent and visualized in a Syngene gel documentation system.

Isolated human glomeruli samples were lysis in RIPA buffer, the concentration of VEGF was measured from supernatant after centrifuge by human VEGF QuantiGlo ELISA Kit (R&D Systems). The concentration of mouse VEGF production in the cell culture medium was measured by mouse VEGF Quantikine ELISA Kit (R&D systems).

Optional whole blood (7 mLx2) was collected from consenting patients at the following time points: pre-dose baseline, 24-48 hours after the day 1 cycle infusion, and at the end of cycle 1. Plasma levels of VEGF and VEGFR2 in duplicate samples were evaluated using enzyme-linked immunosorbent assay (ELISA) (human VEGF QuantiGlo ELISA Kit recognizing VEGF121 and VEGF165 isoforms and human sVEGF R2/KDR Quantikine ELISA Kit, respectively) (R&D Systems, Minneapolis, MN) following the manufacturer's instructions (lower limit of detection=3.30pg/mL and 4.6pg/mL, respectively).