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|
|S6 Ribosomal Protein Mouse mAb Coated Microwells||96 tests|
|S6 Ribosomal Protein Rabbit Detection mAb||1 ea||Green (Lyophilized)|
|Anti-rabbit IgG, HRP-linked Antibody (ELISA Formulated)||1 ea||Red (Lyophilized)|
|Detection Antibody Diluent||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® Total S6 Ribosomal Protein Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of total S6 ribosomal protein. An S6 Ribosomal Protein Mouse mAb has been coated onto the microwells. After incubation with cell lysates, both phospho- and nonphospho-S6 ribosomal proteins are captured by the coated antibody. Following extensive washing, S6 Ribosomal Protein Antibody is added to detect phospho- and nonphospho-S6 ribosomal proteins. HRP-linked Anti-rabbit Antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of optical density is proportional to the quantity of total ribosomal protein.
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® Total S6 Ribosomal Protein Sandwich ELISA Kit detects endogenous levels of S6 ribosomal protein. As shown in Figure 1, a significant induction of phospho-S6 ribosomal protein (Ser235/236) in PDGF treated NIH/3T3 cells is detected. However, the level of 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).
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