![]() ![]() Please see pack insert for specific detergents/protocol modifications needed. Dot Blot protocol technique for detecting, analyzing, and identifying proteins, similar to the western blot technique but differing in that protein samples are not separated electrophoretically but are spotted through circular templates directly onto the membrane or paper substrate. Wash the membrane in washing buffer (3 x 10 min). After incubation, incubate (rotate or shake) the membrane with primary antibody solution (diluted in blocking solution), for 2 hours at room temperature. When dry, incubate the membrane in blocking solution for 1 hour. Note: optimization may be required with IRDye 680LT. Pipette 2l from each fraction onto the membrane, allow the membrane. Once established near-infrared protocols are optimized with IRDye 680RD, IRDye 680LT can be used to optimize signals in the 700 channel. Dilution range 1:20,000 – 1:40,000. Choose IRDye 680LT secondary antibodies to get high signal and for specific uses of detection in the 700nm channel. These antibodies are not recommended when getting up and running on system.Choose IRDye 680RD secondary antibodies to get low background and for general use when detecting in the 700nm channel. Start using IRDye 680RD first over other 700 nm dyes. Dilution working range 1:10,000 – 1:40,000.Choose IRDye 800CW secondary antibodies to get low background and high sensitivity in the 800nm channel. Use IRDye 800CW for targets where your require the highest sensitivity. Dilution working range 1:10,000 – 1:40,000. Optimization guidelines have been developed to enable users to convert the antibody concentrations used in standard immunodetection assays into concentrations.Here are some general guidelines to help you choose the right secondary antibodies: Box 2: Rinse with 1X PBS-T (0.1 Tween 20). Box 1: Rinse with 1X TBS-T (0.1 Tween® 20). Wash blots by shaking vigorously on platform shaker at room temperature for 5 minutes. Select the right IRDye ® or VRDye™ secondary antibody for your application using information in the table below. Wash each blot with a washing buffer that matches the buffer system used for blocking.
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