Revision 4
#7038
Store at +4C
PathScan® Total p70 S6 Kinase Sandwich ELISA Kit
1 Kit
(96 assays)
Species Cross Reactivity:
H M
UniProt ID:
#P23443
Entrez-Gene Id:
#6198
877-616-CELL (2355)
877-678-TECH (8324)
3 Trask Lane | Danvers | Massachusetts | 01923 | USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Product Includes | Product # | Quantity | Color | Storage Temp |
---|---|---|---|---|
p70 S6 Kinase Rabbit Antibody Coated Microwells | 77854 | 96 tests | +4C | |
p70 S6 Kinase Rabbit Detection mAb (Biotinylated) | 14245 | 1 ea | Green (Lyophilized) | +4C |
HRP-Linked Streptavidin (ELISA Formulated) | 11805 | 1 ea | Red (Lyophilized) | +4C |
Detection Antibody Diluent | 13339 | 11 ml | Green | +4C |
HRP Diluent | 13515 | 11 ml | Red | +4C |
TMB Substrate | 7004 | 11 ml | +4C | |
STOP Solution | 7002 | 11 ml | +4C | |
Sealing Tape | 54503 | 2 ea | +4C | |
ELISA Wash Buffer (20X) | 9801 | 25 ml | +4C | |
ELISA Sample Diluent | 11083 | 25 ml | Blue | +4C |
Cell Lysis Buffer (10X) | 9803 | 15 ml | -20C |
*The microwell plate is supplied as 12 8-well modules - Each module is designed to break apart for 8 tests.
Description
The PathScan® Total p70 S6 Kinase Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of p70 S6 kinase. A p70 S6 kinase rabbit antibody has been coated onto the microwells. After incubation with cell lysates, p70 S6 kinase is captured by the coated antibody. Following extensive washing, a biotinylated p70 S6 kinase rabbit detection mAb is added to detect the captured p70 S6 kinase. HRP-linked streptavidin 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 present.
*Antibodies in this kit are custom formulations specific to kit.
Specificity/Sensitivity
The PathScan® Total p70 S6 Kinase Sandwich ELISA Kit detects endogenous levels of p70 S6 kinase, 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.
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).
Background References
- Pullen, N. and Thomas, G. (1997) FEBS Lett 410, 78-82.
- Dufner, A. and Thomas, G. (1999) Exp Cell Res 253, 100-9.
- Weng, Q.P. et al. (1998) J Biol Chem 273, 16621-9.
- Pullen, N. et al. (1998) Science 279, 707-10.
- Alessi, D.R. et al. (1998) Curr Biol 8, 69-81.
- Polakiewicz, R.D. et al. (1998) J Biol Chem 273, 23534-41.
- Fingar, D.C. et al. (2002) Genes Dev 16, 1472-87.
- Saitoh, M. et al. (2002) J Biol Chem 277, 20104-12.
Cross-Reactivity Key
H: Human M: Mouse R: Rat Hm: Hamster Mk: Monkey Vir: Virus Mi: Mink C: Chicken Dm: D. melanogaster X: Xenopus Z: Zebrafish B: Bovine Dg: Dog Pg: Pig Sc: S. cerevisiae Ce: C. elegans Hr: Horse GP: Guinea Pig Rab: Rabbit G: Goat All: All Species Expected
Trademarks and Patents
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
PathScan is a registered trademark of Cell Signaling Technology, Inc.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.
Limited Uses
Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.
Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.
Revision 4
Figure 1. Treatment of MCF7 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 as detected by PathScan® Total p70 S6 Kinase Sandwich ELISA Kit #7038. MCF7 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.
Figure 2. The relationship between lysate protein concentration from untreated and IGF-1-treated MCF7 cells and the absorbance at 450 nm using the PathScan® Total p70 S6 Kinase Sandwich ELISA Kit #7038 is shown. MCF7 cells were treated with 100 ng/ml IGF-1 #3093 for 20 minutes at 37ºC and then lysed.