VisULite ECL Western Blotting Substrate

Detection of GAPDH in HeLa cell lysates visualized with VisULiteTM ECL Western Blotting Substrate using X-ray film or CCD imaging system. A two-fold dilution series of HeLa whole cell lysate was prepared and loaded at 2.5, 1.25, 0.625, 0.313, 0.156, 0.078, 0.039 and 0.019 µg per lane. Samples were transferred onto PVDF membrane and probed with 0.1 µg/mL of Mouse Anti-Human/Mouse/Rat GAPDH Monoclonal Antibody (R&D Systems®, Catalog # MAB5718) followed by HRP-conjugated Anti-Mouse IgG Secondary Antibody (R&D Systems®, Catalog # HAF018). VisULite™ ECL substrate was applied for 1 minute, followed by exposure to a standard sensitivity film (left) or image capture using ProteinSimple® FluorChem™ M (right). Lanes are visualized on a 5 second exposure (film) or 30 second exposure (imager).

Detection of Recombinant Human CRP visualized with VisULite™ ECL Western Blotting Substrate. A two-fold dilution series was prepared using rhCRP (R&D Systems®, Catalog # 1707-CR) and loaded at 200, 100, 50, 25, 12.5, 6.25, 3.13, 1.56 and 0.78 pg per lane. Samples were transferred onto PVDF membrane and probed with 0.5 µg/mL of Sheep Anti-Human CRP (R&D Systems®, Catalog # AF1707) followed by HRP-conjugated Anti-Sheep IgG Secondary Antibody (R&D Systems®, Catalog # HAF016). VisULite™ ECL substrate was applied for 1 minute, followed by exposure to a standard sensitivity film for 30 seconds.

Signal duration with VisULite™ ECL Western Blotting Substrate. A two-fold dilution series of HeLa whole cell lysate was prepared, loaded, transferred and probed with Mouse Anti-Human/Mouse/Rat GAPDH Monoclonal Antibody (R&D Systems®, Catalog # MAB5718) as in Figure 1 (above). VisULiteTM ECL substrate was applied for 1 minute to the PVDF membrane, followed by exposure to a standard sensitivity film for 1 minute at the indicated timepoints. All lanes are visualized on a 1 minute exposure 0 to 30 minutes after VisULite™ ECL substrate addition to the PVDF membrane.