Description
Discover our Western Blotting: tips and troubleshooting eLearning course . Learn approaches to ensure high-quality western blotting courses and receive a certificate upon course completion. Choose between two quick labeling protocols Amersham QuickStain is based on Cy5 NHS ester labeling of reactive amino groups in proteins. First, dilute protein samples 10-fold in the optimized labeling buffer. Then, add a fixed amount of Cy5. Incubate for 5 min at 95°C for qualitative analysis or 30 min at room temperature for quantitative applications. Detect proteins directly after electrophoresis or blotting You do not need to stain and destain your gel for SDS-PAGE analysis. And you can avoid Ponceau S staining of your blot for Western blotting analysis. For either application, simply detect proteins using a fluorescence scanner such as Amersham Typhoon scanner or a CCD imager such as Amersham Imager 600RGB. Comparison with Coomassie and silver staining Amersham QuickStain gives substantially better signal-to-noise and signal-to-background ratios compared with Coomassie staining. It is well suited for purity analysis during protein purification, because it has the sensitivity of silver staining but a wider dynamic range to quantitiate both weak and strong signals. Total protein detection for Western blot normalization With Amersham QuickStain, you can avoid the use of loading controls. Use Cy5 to detect total protein content of a sample and a second dye to detect your protein of interest. This multiplexing capability allows you to evaluate the transfer efficiency and adjust for loading errors using normalization . Fig 1. Multiplex detection of total protein and target protein with Amersham QuickStain (Cy5), Amersham ECL Plex (Ab-Cy3)
