新技术:In-Cell Western Assay-基本实验技术-生物在线 Lab-on-Web

来自 : www.bioon.com.cn/doc/showartic 发布时间：2021-03-25

96 well format Nunc (Part Number 161093, 165305)
96 well format Falcon (Part Number 353075, 353948)

384 well format Nunc (Part Number 164688, 164730)
384 well format Falcon (Part Number 353961, 353962)

The Odyssey Imager requires that microplates have a maximum 4.0 mm distance from the Odyssey scanning surface to the target detection area of the plate. When using the plates specified above for In-Cell Western assays, the recommended focus offset is 3.0 mm. If you use plates other than those recommended above, the focus offset can be determined by scanning a plate containing experimental and control samples at 0.5, 1.0, 2.0, 3.0, and 4.0 mm focus offsets. Use the same intensity settings for each scan. After reviewing the collected scans, use the focus offset with the highest signal-to-noise as your focus offset for experiments. Protect plates from light before imaging to ensure highest sensitivity. When storing plates after imaging, the plates should remain protected from light at room temperature or 4 °C. Intensity for both 700 and 800 nm channels should be set to 5 for initial scanning. If your image signal is saturated or too high, re-scan using a lower intensity setting (i.e., 2.5). If your image signal is too low, re-scan using a higher intensity setting (i.e., 7.5). Scan settings of medium to lowest quality, with 169 m resolution, provide satisfactory results with minimal scan time. Higher scan quality or resolution may be used, but scan time will increase. Establish the specificity of your primary antibody by screening lysates through Western blotting and detection on the Odyssey instrument. If significant non-specific banding is present, choose alternative primary antibodies. Non-specific binding of primaries will complicate interpretation of In-Cell Western assay results. IV. Experimental Results