Akt (PKB, Rac kinase) is a 60kDa ser/thr kinase critical for controlling diverse cellular functions, including glucose metabolism, gene transcription, cell proliferation, and apoptosis. Akt phosphorylates a number of substrates including MBP, glycogen synthetase, PKA RII subunit, and histone H1. Akt is activated in response to insulin and growth factors in a PI3-kinase dependent manner. Activation of PI3-Kinase generates phosphatidylinositol 3,4-bisphosphate, which induces membrane translocation of Akt coincident with its phosphorylation at Thr-308 and Ser-473. Upon activation, Akt associates with members of the PKC family of kinases, such as PKCδ and PKCζ. Ceramide-activated PKCζ leads to phosphorylation of Thr-34 within the pleckstrin homology domain of Akt. This phosphorylation inhibits PIP3 binding to Akt preventing activation of the kinase and may lead to cermide-induced cell death.
References
Western blot analysis of A431 cells untreated (lanes 1 & 3) or treated with 100 ng/ml EGF for 60 min. (lanes 2 & 4). The blots were probed with monoclonal anti-phospho-Akt (Ser-473) (lanes 1 & 2) and monoclonal anti-Akt1 (N-terminal region) (lanes 3 &4).
Western blot analysis of A431 cells, serum starved overnight (20 µg/lanes 1 & 3) and calyculin A (100 nM) treated for 30 minutes (20 µg/lanes 2 & 4). The blot was probed with anti-Akt (Thr-34) (lanes 1 & 2) or anti-Akt1 (N-terminal region) (lanes 3 & 4).
The products are are safely shipped at ambient temperature for both domestic and international shipments. Each product is guaranteed to match the specifications as indicated on the corresponding technical data sheet. Please store at -20C upon arrival for long term storage.
*All molecular weights (MW) are confirmed by comparison to Bio-Rad Rainbow Markers and to western blotmobilities of known proteins with similar MW.
Product References:
RS3251 Kawasaki, H. et al. (2013) World J Gastroenter. 19(17):2629. WB, ICC: mouse intestinal myofibroblastsAM1141 Wu, C. et al. (2012) Cell. 148(5):973. WB: mouse embryonic fibroblastsAM1141 Paterniti, I. et al. (2011) Eur J Neurosci. 33(8):411. WB: mouse spinal cordMS3001 Estrada-Bernal, A. et al. (2011) J Neurooncol. 102:353. Western BlotRS3251 Estrada-Bernal, A. et al. (2011) J Neurooncol. 102:353. Western BlotAM1011 Wei, H., Vander Heide, R. (2010) AJP Heart Circ. 298:H152. WB: rat myocardiumAM1011 Chambers, M.A. et al. (2009) J Physiol. 587:3363. WB: mouse muscle, C2C12AM1011 Moylan, J.S. et al. (2008) AJP Cell. Phys. 295:986. WB: mouse C2C12This kit contains:
| CATALOG# | DESCRIPTION | SIZE | APPLICATIONS | SPECIES REACTIVITY | MW (kDa) |
AM1011 | Akt (N-terminal region) Mouse mAb | 50 μl | WB, E, ICC | Hu, Rt, Ms | 60 |
AM1141 | Akt (Ser-473), phospho-specific Mouse mAb | 50 μl | WB, E, IP | Hu, Rt, Ms | 60 |
AP1001 | Akt (Thr-34), phospho-specific Rabbit pAb | 50 μl | WB, E | Hu, Rt, Rb | 60 |
MS3001 | Anti-Mouse Ig:HRP Donkey pAb | 100 μl | WB, E | Ms | |
RS3251 | Anti-Rabbit Ig Light-Chain Specific:HRP Mouse mAb | 100 μl | WB, E, ICC, IHC | Rb |
KIT SUMMARY
The Akt phospho-regulation antibody sampler kit can be used to detect Akt activation via Ser-473 phosphorylation, as well as Akt inhibition through Thr-34 phosphorylation. The kit also includes a monoclonal antibody to monitor the total Akt expression levels and secondary reagents for detection of these antibodies.
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我现在做关于细胞膜分子交联，然后用western检测多聚体的实验
遇到很大的困难
刚开始用真核细胞做，就发现细胞交联后提取蛋白，比未交联的细胞蛋白总量减少
后来用原核先提取蛋白做实验
发现交联终止后，做western交联的孔曝光没有条带，后来用同样的方法重新做了一次PAGE，染胶，发现蛋白不见了
为什么交联后蛋白减少或是不见了呢
我用的都是Pierce公司的交联剂，用过EGS和BOSCOES，都有同样的问题
方法是综合说明书和一片文献上来的
到底问题出在哪里呢
请各位大侠帮帮忙
实验卡在这里进行不下去了
我快急死了
请有经验的，用过交联剂的老师，同学多多指导
万分感谢！！
明胶与干酪素都是蛋白质类，其分子结构同属氨基酸，但前者比后者价格便宜很多，而且在我国的产盆很大，因此具有代替干酪素做涂料胶粘剂的可能性。
高分子化合物是指那些由众多原子或原子团主要以共价键结合而成的相对分子量在一万以上的化合物，由千百个原子彼此以共价键结合形成相对分子质量特别大、具有重复结构单元的化合物(可分为无机高分子化合物和有机高分子化合物）。
是由一类相对分子质量很高的分子聚集而成的化合物，也称为高分子、大分子等。大多数高分子的相对分子质量在一万到百万之间，其分子链是由许多简单的结构单元通过共价键重复连接而成。一般把相对分子质量高于10000的分子称为高分子。高分子通常由10^3～10^5个原子以共价键连接而成。由于高分子多是由小分子通过聚合反应而制得的，因此也常被称为聚合物或高聚物，用于聚合的小分子则被称为“单体”。
举例：纤维素、蛋白质、蚕丝、橡胶、淀粉等天然高分子化合物，以及以高聚物为基础的合成材料，如各种塑料，合成橡胶，合成纤维、涂料与粘接剂等。
同一种高分子化合物的分子链所含的链节数并不相同，所以高分子化合物实质上是由许多链节结构相同而聚合度不同的化合物所组成的混合物，其相对分子质量与聚合度都是平均值。
高分子化合物几乎无挥发性，常温下常以固态或液态存在。固态高聚物按其结构形态可分为晶态和非晶态。前者分子排列规整有序；而后者分子排列无规则。同一种高分子化合物可以兼具晶态和非晶态两种结构。大多数的合成树脂都是非晶态结构。
高分子的分子结构基本上只有两种，一种是线型结构，另一种是体型结构。线型结构的特征是分子中的原子以共价键互相连结成一条很长的卷曲状态的“链”（叫分子链）。体型结构的特征是分子链与分子链之间还有许多共价键交联起来，形成三度空间的网络结构。这两种不同的结构，性能上有很大的差异。
从交联剂的配制，以及交联反应体系，交联后样品是否沸水煮10min,最后SDS-PAGE检测。
P酸+5碳糖+含氮碱基形成脱氧核酸【DNA】,通过转录+翻译【涉及到的有转移RNA,信使RNA】然后识别信使RNA上的碱基排列顺序,把密码子连接起来,同时转移RNA上核糖体脱落形成氨基酸,氨基酸的排列顺序
