We will develop a system for the microdeposition of multiple phosphorylation site-specific antibodies onto protein bands of 1D or spots of 2D SDS-PAGE blots in the performance of protein phosphorylation studies. We will design and build a low volume loading print head for microdeposition of up to 10 different phosphospecific antibodies, or other fluids, pipette loaded at 3-5 mu L per channel. We will demonstrate the use of the low volume print head to deposit phosphospecific antibodies onto non-muscle Myosin Heavy Chain (MHC) bands in a Western blot to investigate the serine and threonine phosphorylation of MHC in mast cells during the antigen-induced secretory process. This new method has the significant advantage in examining for the presence and specific phosphorylation status of many proteins contained within one protein blot. This process will be more rapid, consume significantly less antibody, require a fewer number of gels and blots, and conserve precious protein samples. In circumstances where a protein sample is limited or the source of the antibody is rare or expensive, the Micro-Western blot method will provide a powerful alternative to conventional Western blotting methods. ? ?
