Relationship between protein concentration of lysates from untreated and IGF-1-treated MCF7 cells and immediate light generation with chemiluminescent substrate is shown. Cells were treated with IGF-1 #3093 (100 ng/ml) and lysed after incubation at 37ºC for 20 minutes. Graph inset corresponding to the shaded area shows high sensitivity and a linear response at the low protein concentration range.
The PathScan® Phospho-p70 S6 Kinase (Thr389) Chemiluminescent Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of phospho-p70 S6 kinase (Thr389) protein with a chemiluminescent readout. Chemiluminescence ELISAs often have a wider dynamic range and higher sensitivity than conventional chromogenic detection. This chemiluminescent ELISA which is offered in low volume microplates, shows increased signal and sensitivity while using a smaller sample size. A p70 S6 kinase rabbit antibody has been coated on the microwells. After incubation with cell lysates, both phospho- and nonphospho-p70 S6 kinase proteins are captured by the coated antibody. Following extensive washing, phospho-p70 S6 kinase (Thr389) mouse antibody is added to detect the captured phospho-p70 S6 kinase (Thr389) protein. Anti-mouse IgG, HRP-linked antibody is then used to recognize the bound detection antibody. Chemiluminescent reagent is added for signal development. The magnitude of light emission, measured in relative light units (RLU), is proportional to the quantity of phospho-p70 S6 kinase (Thr389) protein.
Antibodies in kit are custom formulations specific to kit.
PathScan® Phospho-p70 S6 Kinase (Thr389) Chemiluminescent Sandwich ELISA Kit #7153 detects endogenous levels of phospho-p70 S6 kinase (Thr389) in human and mouse cells. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
p70 S6 kinase is a mitogen activated Ser/Thr protein kinase that is required for cell growth and G1 cell cycle progression (1,2). p70 S6 kinase phosphorylates the S6 protein of the 40S ribosomal subunit and is involved in translational control of 5' oligopyrimidine tract mRNAs (1). A second isoform, p85 S6 kinase, is derived from the same gene and is identical to p70 S6 kinase except for 23 extra residues at the amino terminus, which encode a nuclear localizing signal (1). Both isoforms lie on a mitogen activated signaling pathway downstream of phosphoinositide-3 kinase (PI-3K) and the target of rapamycin, FRAP/mTOR, a pathway distinct from the Ras/MAP kinase cascade (1). The activity of p70 S6 kinase is controlled by multiple phosphorylation events located within the catalytic, linker and pseudosubstrate domains (1). Phosphorylation of Thr229 in the catalytic domain and Thr389 in the linker domain are most critical for kinase function (1). Phosphorylation of Thr389, however, most closely correlates with p70 kinase activity in vivo (3). Prior phosphorylation of Thr389 is required for the action of phosphoinositide 3-dependent protein kinase 1 (PDK1) on Thr229 (4,5). Phosphorylation of this site is stimulated by growth factors such as insulin, EGF and FGF, as well as by serum and some G-protein-coupled receptor ligands, and is blocked by wortmannin, LY294002 (PI-3K inhibitor) and rapamycin (FRAP/mTOR inhibitor) (1,6,7). Ser411, Thr421 and Ser424 lie within a Ser-Pro-rich region located in the pseudosubstrate region (1). Phosphorylation at these sites is thought to activate p70 S6 kinase via relief of pseudosubstrate suppression (1,2). Another LY294002 and rapamycin sensitive phosphorylation site, Ser371, is an in vitro substrate for mTOR and correlates well with the activity of a partially rapamycin resistant mutant p70 S6 kinase (8).
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Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
PathScan is a trademark of Cell Signaling Technology, Inc.
U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.