PathScan Sandwich ELISA Kits

PathScan® Phospho-p70 S6 Kinase (Thr389) Sandwich ELISA Kit #7063

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Applications Key:
Reactivity Key: H = Human  M = Mouse  
Species cross-reactivity is determined by Western blot.

Protocols

7063

Sandwich ELISA,

Specificity / Sensitivity

The PathScan® Phospho-p70 S6 Kinase (Thr389) Sandwich ELISA Kit detects endogenous levels of p70 S6 kinase when phosphorylated at Thr389, as shown in Figure 1. This kit is predicted to cross-react with the p85 isoform of S6 kinase. The kit sensitivity is shown in Figure 2.

Description

The PathScan® Phospho-p70 S6 Kinase (Thr389) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of phospho-p70 S6 kinase phosphorylated at Thr389. A p70 S6 kinase rabbit mAb has been coated onto the microwells. After incubation with cell lysates, p70 S6 kinase is captured by the coated antibody. Following extensive washing, a phospho-p70 S6 kinase (Thr389) mouse detection mAb is added to detect the captured phospho-p70 S6 kinase (Thr389). Anti-mouse IgG, HRP-linked antibody is then used to recognize the bound detection antibody. The HRP substrate TMB is added to develop color. The magnitude of the absorbance for this developed color is proportional to the quantity of p70 S6 kinase phosphorylated at Thr389.

ELISA - Western correlation

Figure 1. Treatment of MCF-7 cells with IGF-1 stimulates phosphorylation of p70 S6 kinase at Thr389 as detected by the PathScan® Phospho-p70 S6 Kinase (Thr389) Sandwich ELISA Kit #7063, but does not affect the level of total p70 S6 kinase. MCF-7 cells were treated with 100 ng/ml IGF-1 #3093 for 20 minutes at 37ºC. The absorbance readings at 450 nm are shown in the top figure, while corresponding western blots using p70 S6 Kinase Antibody #9202 (left panel) and Phospho-p70 S6 Kinase (Thr389) (1A5) Mouse mAb #9206 (right panel) are shown in the bottom figure.

Sensitivity

Figure 2. The relationship between lysate protein concentration from untreated and IGF-1-treated MCF-7 cells and the absorbance at 450 nm using the PathScan® Phospho-p70 S6 Kinase (Thr389) Sandwich ELISA Kit #7063 is shown. MCF-7 cells were treated with 100 ng/ml IGF-1 #3093 for 20 minutes at 37ºC and then lysed.

Background

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).

1. Pullen, N. and Thomas, G. et al. (1997) FEBS Lett.410, 78 - 82.
2. Dufner, A. and Thomas, G. et al. (1999) Exp. Cell Res.253, 100 - 109.
3. Weng, Q.P. et al. (1998) J. Biol. Chem.273, 16621 - 16629.
4. Pullen, N. et al. (1998) Science279, 707 - 710.
5. Alessi, D.R. et al. (1998) Curr. Biol.8, 69 - 81.
6. Polakiewicz, R.D. et al. (1998) J. Biol. Chem.273, 23534 - 23541.
7. Fingar, D.C. et al. (2002) Genes Dev.16, 1472 - 1487.
8. Saitoh, M. et al. (2002) J. Biol. Chem.277, 20104 - 20112.

Application References

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Companion Products

• 7004 TMB Substrate
• 9206 Phospho-p70 S6 Kinase (Thr389) (1A5) Mouse mAb
• 9234 Phospho-p70 S6 Kinase (Thr389) (108D2) Rabbit mAb
• 2708 p70 S6 Kinase (49D7) Rabbit mAb
• 7002 STOP Solution
• 9808 Phosphate Buffered Saline (PBS-20X)
• 9809 Phosphate Buffered Saline with Tween 20 (PBST-20X)

This product is for in vitro research use only and is not intended for use in humans or animals. This product is not intended for use as therapeutic or in diagnostic procedures.