The Pathscan® Multiplex Western Cocktail I Detection Kit offers a unique method to assay the activation of multiple pathways on one membrane without stripping and reprobing. This method saves the user valuable time, while increasing accuracy and minimizing reagent waste. The system allows the user to simultaneously detect levels of phospho-p90RSK, phospho-Akt, phospho-p44/42 MAPK (Erk1/2) and phospho-S6 ribosomal protein. The kit also includes Rab11 antibody to control protein loading. In addition, each Pathscan® Multiplex Western Cocktail Detection Kit contains treated and untreated cell lysates and the Phototope®-HRP Western Detection System. The kit includes enough primary and secondary antibodies to perform five Western blot experiments.
Each phospho-antibody in this kit recognizes only the phosphorylated form of its specific target. The Rab11 antibody detects total levels of target protein to control for protein loading. All the antibodies detect endogenous levels of target proteins.
Antibodies are produced by immunizing animals with synthetic peptides. Polyclonal antibodies are purified by protein A and peptide affinity chromatography.
Akt, also referred to as PKB or Rac, plays a critical role in controlling the balance between survival and apoptosis (1-3). This protein kinase is activated by insulin and various growth and survival factors, and functions in a wortmannin-sensitive pathway involving PI3 kinase (2,3). Akt is activated by phospholipid binding and activation loop phosphorylation at Thr308 by PDK1 (4) and also by phosphorylation within the carboxy-terminus at Ser473.
Both p44 and p42 MAP kinases (Erk1 and Erk2) play a critical role in the regulation of cell growth and differentiation (5-8). MAP kinases are activated by a wide variety of extracellular signals including growth and neurotrophic factors, cytokines, hormones and neurotransmitters. Activation of MAP kinases occurs through phosphorylation of threonine and tyrosine (202 and 204 of human MAP kinase [Erk1] or 183 and 185 of rat Erk2) at the sequence T*EY* by a single upstream MAP kinase kinase (MEK) (9,10). One of the downstream targets of p44/42 MAPK is p90RSK.
To effectively promote growth and cell division in a sustained manner, growth factors and mitogens must upregulate translation (11,12). Growth factors and mitogens induce the activation of p70 S6 kinase, which in turn phosphorylates the S6 ribosomal protein. Phosphorylation of S6 correlates with an increase in translation, particularly of mRNAs with an oligopyrimidine tract in their 5+ untranslated regions (12).
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