研究生分子生物学工作基础新基因研究路线.pptVIP

Immunoprecipitation Co-Immnunoprecipitation (co-IP) Co-IP Optimization Strategies Complex binding Use lysis and wash buffers with low ionic strength (i.e., 120mM NaCl) that contain non-ionic detergents (NP-40 and Triton X-100), which are less likely to disrupt protein-protein interactions. lysing cells by sonication or vortexing the lysate or bead-bound immune complexes during the wash steps should be avoided to prevent the disruption of the protein-protein interaction(s) of the target complex. Crosslinking the binding partners to strengthen protein-protein interactions. High background from nonspecific interactions Changing the ionic strength of the IP buffer by titrating the salt concentration from 120 to 1000mM. Decreasing the amount of primary antibody until the signal:noise ratio is maximized. Preclearing the lysates. Antibody Antibody quality crosslinking antibody to Protein A/G-coated beads or covalently binding antibody directly to treated beads. GST pull-down /resources/product-guides-and-selectors/protocols-and-applications-guide/protein-expression/ Fluorescent Imaging a-CBP Stealth? RNAi　　—最有效进行基因沉默的RNAi双链体 　　　　Stealth RNAi是经化学修饰合成的RNAi双链体，可产生高特异性的有效的基因表达抑制。它克服了非修饰　 　siRNA分子的限制，因此可以产生更清晰的结果。 　　。避免脱靶效应—Stealth RNAi的正义链是经过化学修饰的，仅反义链能够可进入RNAi通路，解决了敏感　　　　　　　　　的脱靶问题。。血清和细胞培养中的高稳定性—在进行动物研究时无需验证新的试剂，节约您的时间。。消除非特异性效应—避免了细胞应激反应通路的激活，避免产生非特异结果。 * Many proteins function with partners or as components of large multi-protein complexes. Understanding these interactions is critical to understanding biological pathways and cellular function. Glutathione-S Transferase (GST) pull-down (1) is an important tool for validation of suspected protein:protein interactions and for identification of interacting partners (2–5). In GST pull-down assays a GST-fusion bait protein is expressed in E. coli, then bound to glutathione (GSH)-coupled particles and used to affinity purify any proteins (prey) that interact with the bait from a pool of proteins in solution (