Figure 1: Treatment of NIH/3T3 cells with PDGF stimulates phosphorylation of S6 ribosomal protein at Ser235/236, detected by PathScan® Phospho-S6 Ribosomal Protein (Ser235/236) Sandwich ELISA Kit #7205, but does not affect the level of total S6 ribosomal protein detected by PathScan® Total S6 Ribosomal Protein Sandwich ELISA Kit #7225. A combination of rapamycin, a FRAP/mTOR inhibitor, U0126, a MEK1/2 inhibitor, and LY294002, a PI3 Kinase inhibitor, can totally suppress the phosphorylation of S6 ribosomal protein in cells. Triple inhibitor-treatment (37ºC for 120 min after starvation) represents the nonphosphorylated form of S6 ribosomal protein whereas PDGF-treated cells represent the phosphorylated form as shown in both Sandwich ELISA and Western analysis (upper/bottom, right). OD450 readings are shown in the top figure, while the corresponding Western blot, using Phospho-S6 Ribosomal Protein (Ser235/236) Ab #2211 (right panel) or S6 Ribosomal Protein Antibody #2212 (left panel), is shown in the bottom figure.
Figure 2: The relationship between protein concentration of lysates from triple inhibitor-treated (as control) and PDGF-treated NIH/3T3 cells and kit assay optical density readings is shown. NIH/3T3 cells (70-85% confluence) were treated with PDGF (50 ng/ml), and lysed after incubation at 37ºC for 30 min. For the control, NIH/3T3 cells (70-85% confluence) were treated with rapamycin (100 nM), U0126 (10 μM), and LY294002 (50 µM), and lysed after incubation at 37ºC for 120 min.
|Product Includes||Volume||Solution Color|
|Phospho-S6 Ribosomal Protein (Ser235/236) Rabbit mAb Coated Microwells||96 tests|
|S6 Ribosomal Protein Mouse Detection mAb||1 ea||Green (Lyophilized)|
|Anti-mouse IgG, HRP-linked Antibody (ELISA Formulated)||1 ea||Red (Lyophilized)|
|Detection Antibody Diluent 3||11 ml||Green|
|HRP Diluent||11 ml||Red|
|TMB Substrate 7004||11 ml|
|STOP Solution 7002||11 ml|
|Sealing Tape||2 ea|
|ELISA Wash Buffer (20X) 9801||25 ml|
|ELISA Sample Diluent||25 ml||Blue|
|Cell Lysis Buffer (10X) 9803||15 ml|
The PathScan® Phospho-S6 Ribosomal Protein (Ser235/236) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of phospho-S6 ribosomal protein (Ser235/236). A Phospho-S6 Ribosomal Protein (Ser235/236) Antibody has been coated onto the microwells. After incubation with cell lysates, only phospho-S6 ribosomal protein is captured by the coated antibody. Following extensive washing, a Total S6 Ribosomal Protein Mouse mAb is added to detect the captured phospho-S6 ribosomal protein (Ser235/236). Anti-Mouse IgG, HRP-linked Antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of optical density for this developed color is proportional to the quantity of phospho-S6 ribosomal protein (Ser235/236).
Antibodies in kit are custom formulations specific to kit.
NOTE: Prepare solutions with purified water.
*NOTE: Some PathScan® ELISA Kits may include HRP-Linked Streptavidin in place of HRP-Linked Antibody.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.
posted November 2013
Protocol Id: 204
The PathScan® Phospho-S6 Ribosomal Protein (Ser235/236) Sandwich ELISA Kit detects endogenous levels of phospho-S6 ribosomal protein phosphorylated on serines 235/236. As shown in Figure 1, a significant induction of phospho-S6 ribosomal protein (Ser235/236) in PDGF treated NIH/3T3 cells is detected by this kit. However, total S6 ribosomal protein (phospho and non-phospho) remains unchanged. NIH/3T3 or 293 cells treated with 20% FBS after starvation also show similar results (data not shown). This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
One way that growth factors and mitogens effectively promote sustained cell growth and proliferation is by upregulating mRNA translation (1,2). Growth factors and mitogens induce the activation of p70 S6 kinase and the subsequent phosphorylation of the S6 ribosomal protein. Phosphorylation of S6 ribosomal protein correlates with an increase in translation of mRNA transcripts that contain an oligopyrimidine tract in their 5' untranslated regions (2). These particular mRNA transcripts (5'TOP) encode proteins involved in cell cycle progression, as well as ribosomal proteins and elongation factors necessary for translation (2,3). Important S6 ribosomal protein phosphorylation sites include several residues (Ser235, Ser236, Ser240, and Ser244) located within a small, carboxy-terminal region of the S6 protein (4,5).
Explore pathways + proteins related to this product.
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's 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's 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.