做Western-blot仪器Odyssey.ppt

Odyssey 2006.ppt * Another application used with increasing frequency by our Odyssey and Aerius customers is the in-cell western or cell based assay. The ICW is a high-throughput assay used to detect and quantify two protein targets directly within cells. How is an ICW performed? A typical workflow is shown here. The assay starts with the transfer and culture of cells in a 96 or 384 well plate. No special plates are required at this point, so the standard plates that you may have in your lab are able to be used. Once the cells have grown to confluency within the plates, they are treated as the experimental procedure dictates. For example, an inhibitor or stimulator may be added. The cells are then fixed with either formaldehyde or a less volatile fixative such as Prefer (an alcohol based fixative). The cell membranes are permeabilized with detergent prior to adding the antibodies. At this stage, there are two options for proceeding: (first click) One option is that two primary antibodies can be added. This enables two protein targets to be detected for two different reasons. First, one target can be used to normalize for the number of cells. An example of this would be using total ERK and phospho ERK primaries. Second, if normalization is not important for the experimental design, then each primary can detect two entirely different targets. Following the addition of the primaries, two secondaries are then added, one each channel, for example IRDye 680 and 800CW. (second click) The second option is that only one primary antibody can be added and a DNA stain can be used for normalization. This is more affordable than using primary antibodies for normalization. Following the addition of the primary antibody, the secondary antibody of choice as added as is the DNA stain – To-Pro-3 for example) (third click) Whichever option is chosen, once the secondary or secondaries are added, the plates are washed and following that, the plate is ready to scan on t