SPR技术、体外转录和翻译、GST融合蛋白、免疫共沉淀、串联亲和纯化等鉴定分析蛋白质之间相互作用介绍.pptVIP

* * The processivity issue of cell-free systems is where the Rapid Translation System provides a solution. Based on the findings of Alexander Spirin, a device was developed in which the reaction chamber is separated from the so-called feeding solution by a semi-permeable membrane. In the reaction chamber all the components for a coupled transcription-translation reaction are present: the DNA template carrying the gene of interest, the T7 RNA polymerase to generate a mRNA molecule from this template and the translation machinery from an E. Coli lysate. In the feeding chamber the amino acids, energy substrates, buffer components, NTPs etc. are present. By a continuous exchange over the membrane waste from the reaction is diluted in the feeding mix and new supply for the translation reaction is provided. Magnets are present to continuously mix the contents of both chambers by placing the device in the reaction controller. Applying this Continuous Exchange Cell Free (CECF) principle allows translation reactions to continue for up to 24 hours resulting in yields of up 500 μg of protein/ml. * SPR技术、体外转录和翻译、GST融合蛋白、免疫共沉淀、串联亲和纯化等鉴定分析蛋白质之间相互作用技术介绍 SPR技术分析蛋白质相互作用 体外转录和翻译鉴定蛋白质相互作用 coupled transcription/translation reaction 偶联的转录和翻译过程 continuous exchange cell-free (CECF) principle 持续交换和非细胞体系的原理 偶联的转录和翻译过程 T7-RNA polymerase E.coli lysate gene DNA coupled transcription / translation protein gene 持续交换和非细胞体系的原理 T7 RNA polymerase Translation machinery E. coli Energy regenerating system Protein DNA template Amino acids Energy substrates Buffer components NTPs Supply Amino acids Energy substrates Buffer components NTPs Supply Inhibitory by-products Inhibitory by-products membrane membrane 供给腔 反应腔 供给腔 microtiter- plate PCR tubes RTS 500 devices microcentrifuge tubes 4x12 RTS 100 8x 96x RTS 100 RTS500 反应装置 reaction chamber feeding chamber with semipermeable membranes RTS 500 ProteoMaster E. coli HY Kit 质谱