| Product Name | Crosstide [GRPRTSSFAEG]GRPRTSSFAEG |
| Size | 5 mg |
| Catalog # | AS-60209-5 |
| US$ | $289 |
| Purity | % Peak Area By HPLC ≥ 95% |
Crosstide is a peptide analog of glycogen synthase kinase-3 (GSK-3) that functions as a natural substrate for Akt/PKB. It displays similar specificities towards PKBα, PKBβ and PKBγ isoforms. It is also recognized by MAPKAP kinase-1 and p70S6K. The fluorescent and biotinlyated peptides demonstrate similar enzyme activities. | |
| Detailed Information | |
| Storage | -20°C |
| References | Ref: Dai, F. et al. Biochem. Biophys. Res. Commun. 293, 1191 (2002). Meier, R. et al. J. Biol. Chem. 272, 30491 (1997); Chan, TO. et al. Ann. Rev. Biochem. 68, 965 (1999). |
| Molecular Weight | 1164.2 |
| GRPRTSSFAEG | |
| Sequence(Three-Letter Code) | H - Gly - Arg - Pro - Arg - Thr - Ser - Ser - Phe - Ala - Glu - Gly - OH |
| Product Citations | Okamura, T. et al. (2009). Tyrosine phosphorylation of the human glutathione S-transferase P1 by epidermal growth factor receptor.J. Biol. Chem. 284, 16979. |
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二、限制性内切酶消化样品DNA
三、凝胶电泳分离消化产物
四、如果靶序列>5kb,在0.25M的HCl中进行震荡脱嘌呤10min,ddH2O洗一次
五、用变性液震荡处理30min,ddH2O洗一次
六、中和液震荡处理30min
七、裁取合适大小的尼龙膜或硝酸纤维素膜进行转膜操作,可用真空转膜仪或者搭滤纸桥,需要20×SSC
八、制备探针,可用地高辛标记(以地高辛为例)
九、将膜放入杂交瓶,42°C预杂交30min
十、倒掉预杂交液,加入杂交液,适当的温度进行杂交4h至过夜
十一、洗膜,先用2×SSC+0.1%SDS,20-25°C洗2×5min,再用0.5×SSC+0.1%SDS洗2×15min。然后用washing buffer洗1-5min,接着用blocking solution洗30min后,用antibody solution洗30min,再用washing buffer洗2×15min,再用detection buffer洗2-5min后,取出膜,放于保鲜膜上,在结合有DNA的一面滴加CSPD ready-to-use后,立刻盖上保鲜膜,让CSPD ready-to-use均匀的布满膜表面,室温放置5min后,37°C温育10min以上
十二、放射自显影,可用成像系统信号累积模式显影或用X-ray显影
应该注意以下2个方面
1 用玻璃棒将气泡赶净 有气泡的位置不导电 蛋白就转不上
2 半干式转膜仪要保证一定量的转移缓冲液,如果转膜一个小时以上,缓冲液要多一些
我用Bio-rda转膜仪,转膜2分钟就停止,然后机器上显示“noloaddetected“。各位知道这是为什么吗??
第一次出现这种情况,同样的条件,之前一直没有问题。

