Download Current Protocols in Chemical Biology 2010 (Volume 2) by Adam P. Arkin PDF

By Adam P. Arkin

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Resuspend beads in 20 μl 1× RIPA buffer containing 1× protease inhibitor, 1× phosphatase inhibitor, and 1× sample buffer for SDS-PAGE. 15. 5 min. 16. Load the samples onto an SDS-PAGE gel and proceed with immunoblotting as described in steps 6 to 8 of Support Protocol 1, using thiophosphate ester rabbit monoclonal antibody. PREPARATION OF A THIOPHOSPHORYLATED POSITIVE CONTROL PEPTIDE AND PROTEIN The two control samples, a thiophosphorylated peptide and protein, can be prepared from easily purchased materials.

10. Optimize printer settings (dwell time, velocity, and number of pre-spots) to achieve uniform spots of the desired size. The amount of material transferred during printing depends on the pin’s weight, composition, and geometry, as well as the slide’s surface properties. Thus, for each combination of pins and slides, printer settings should be optimized to obtain uniform spots of the desired size. As a starting point, the authors’ preferred settings for printing are described in Table 1. The Troubleshooting section discusses strategies for refining printing parameters.

Lectins can be labeled with a fluorophore for direct detection or can be labeled with a tag, such as biotin, followed by detection with an appropriate fluorophore-labeled secondary reagent, such as Cy3-labeled streptavidin. Monoclonal and serum antibodies are typically detected with a fluorophore-labeled secondary antibody specific for the primary antibody’s species and isotype of interest. Selection of an appropriate fluorophore is a key consideration. In addition to matching the appropriate excitation and emission properties for the scanner, degradation and photobleaching should be considered.

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