Micro-Western Blot for Proteomics Studies

描述（由申请人提供）：我们将开发一种系统，用于在蛋白磷酸化研究中将多个磷酸化位点特异性抗体微沉积到1D蛋白条带或2D SDS-PAGE印迹斑点上。我们将设计和构建一个低容量装载打印头，用于最多10种不同磷酸化特异性抗体或其他流体的微沉积，每个通道以3-5 μ L的移液管装载。我们将证明使用低容量打印头存款磷酸化抗体的非肌肉肌球蛋白重链（MHC）带在蛋白质印迹法研究的丝氨酸和苏氨酸磷酸化的MHC在肥大细胞在抗原诱导的分泌过程中。这种新方法在检测一个蛋白质印迹中包含的许多蛋白质的存在和特异性磷酸化状态方面具有显著的优势。这一过程将更加快速，消耗的抗体显著减少，需要的凝胶和印迹数量减少，并保存珍贵的蛋白质样品。在蛋白质样品有限或抗体来源稀少或昂贵的情况下，Micro-Western印迹方法将为常规Western印迹方法提供强有力的替代方案。

DESCRIPTION (provided by applicant): 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.