pCaspase3-sensor信号通路质粒

基本信息

原核抗性:	卡那霉素Kan
筛选标记:	遗传霉素G418
克隆菌株:	大肠杆菌DH5α
培养条件:	37℃

质粒简介

           pCaspase3-sensor是一个哺乳细胞黄色荧光蛋白信号通路报告质粒。pCaspase3-Sensor Vector can be used to detect the onset of caspase-3 activity in mammalian cells. This vector encodes the enhanced yellow-green variant (EYFP) of the Aequorea victoria green fluorescent protein (GFP) fused at the 3' end to three copies of the nuclear localization signal (NLS) of the simian virus 40 large T-antigen (1,2). At the 5' end the gene contains a sequence encoding the nuclear export signal (NES) of the Map Kinase Kinase (MAPKK;3). The NES is separated from the EYFP coding region by a 36-nucleotide sequence encoding the region of Poly (ADP-ribose) polymerase (PARP) cleaved by caspase-3. The complete coding sequence for this fusion protein is human codon-optimized (4). Because the NES of MAPKK dominates the NLS, the full-length fluorescent fusion protein distributes to the cytosol. If caspase-3 is activated, the NES will be cleaved from the fusion protein and the truncated EYFP-NLS fusion will translocate to the nucleus via the NLS. The translocation of the fluorescent protein from the cytosol to the nucleus indicates caspase-3 activity at a cellular level. The fluorescence excitation maximum of EYFP is 513 nm; the emission spectrum has a peak at 527 nm (in the yellow-green region). The Em of EYFP at 513 nm is 36,500 cm–1M–1 and its quantum yield is 0.63 (5), resulting in a bright fluorescent signal. The fluorescence intensity is roughly equivalent to that of EGFP.
          The vector contains an SV40 origin of replication and a neomycin resistance (Neor) gene for selection (using G418) in eukaryotic cells. A bacterial promoter (P) upstream of Neor expresses kanamycin resistance in E. coli. The vector backbone also provides a pUC19 origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.
          The pCaspase3-Sensor Vector can be introduced into mammalian cells using any standard transfection method. If desired, stable transfectants can be selected using G418 (6).pCaspase3-Sensor can be used to detect the onset of caspase-3 activity in mammalian cells. This vector allows visual monitoring of caspase-3 activity in the cell via fluorescence microscopy. If the fluorescent fusion protein encoded by the pCaspase3-Sensor vector is in the cytosol, caspase-3 is not active. However, if caspase-3 becomes active, the dominant nuclear export signal (NES) is cleaved from the fusion protein and the truncated fluorescent protein will become localized to the nucleus via the nuclear localization signal.

质粒图谱

 
pCaspase3-sensor信号通路质粒使用说明：
    
     1、收到质粒干粉后请先5000rpm离心1min，再加入20μl无菌水溶解质粒，室温放置1min；
     2、从-80℃冰箱中取出相应的感受态，置于冰盒上解冻，并做好标记；
     3、取2μl质粒加至100μl感受态中，冰浴30min；
     4、42℃热激90s，再冰浴2min；
     5、加入900μl无抗的LB液体培养基，180rpm震荡培养45min；
     6、6000rpm离心5min，仅留100ul上清混匀菌体沉淀；
     7、混匀后的菌液加至对应抗性的LB平板上，倒入适量玻璃珠，涂匀液体；
     8、将平板正向培养1h，再倒置培养12h~16h；
     9、挑取单克隆菌落至对应抗性的LB液体培养基中，震荡培养12h~16h，根据实验需要提取质粒。
 
pCaspase3-sensor信号通路质粒注意事项：
 
      1、如果您收到的是甘油菌种，请先四区划线，挑取单克隆培养。
      2、如果第二天转化平板长的过多，请将质粒按比例稀释后再转化。