Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® Phospho-S6 Ribosomal Protein (Ser235/236) Sandwich ELISA Kit #7205

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Kit Includes Volume Solution Color
P-S6 Ribosomal Protein (S235/236) AbCoated Microwells
S6 Ribosomal Protein Detection Ab 11 ml Green
Anti-mouse IgG, HRP-linked Antibody 11 ml Red
TMB Substrate #7004 11 ml Colorless
STOP Solution #7002 11 ml Colorless
Sealing Tape 2 sheets
ELISA Wash Buffer (20X) 25 ml Colorless
ELISA Sample Diluent 25 ml Blue
Cell Lysis Buffer (10X) #9803 15 ml Yellowish

Note: 12 8-well modules –Each module is designed to break apart for 8 tests.
Note: Kit should be stored at 4°C with the exception of Cell Lysis Buffer, which is stored at –20°C (packaged separately).

Species Cross-Reactivity

H M

Reactivity Key:  H=Human  M=Mouse
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Description

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). HRP-linked Anti-mouse 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.

Specificity / Sensitivity

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.

ELISA - Western correlation

ELISA - Western correlation

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.

Sensitivity

Sensitivity

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.

Background

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).

  1. Dufner, A. and Thomas, G. (1999) Exp. Cell Res. 253, 100-109.
  2. Peterson, R.T. and Schreiber, S.L. (1998) Curr. Biol. 8, R248-R250.
  3. Jefferies, H.B. et al. (1997) EMBO J. 16, 3693-3704.
  4. Ferrari, S. et al. (1991) J. Biol. Chem. 266, 22770-22775.
  5. Flotow, H. and Thomas, G. (1992) J. Biol. Chem. 267, 3074-3078.

Application References

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For Research Use Only. Not For Use In Diagnostic Procedures.

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