Figure 1: Serum stimulation of starved NIH/3T3 cells with 20% FBS promotes phosphorylation of S6 ribosomal protein at Ser240/244 as detected by PathScan® Phospho-S6 Ribosomal Protein (Ser240/244) Sandwich ELISA Kit #13911, but does not affect the level of total S6 ribosomal protein detected by PathScan® Total S6 Ribosomal Protein Sandwich ELISA Kit #7225. The absorbance readings at 450 nm are shown in the top of the figure while corresponding western blots using S6 Ribosomal Protein (5G10) Rabbit mAb #2217 (left) and Phospho-S6 Ribosomal Protein (Ser240/244) (D68F8) XP® Rabbit mAb #5364 (right) are shown in the bottom of the figure.
Figure 2. The relationship between protein concentration of untreated or 20% FBS-treated lysates and the absorbance at 450 nm as detected by the PathScan® Phospho-S6 Ribosomal Protein (Ser240/244) Sandwich ELISA Kit is shown. NIH/3T3 cells were cultured (85% confluence), starved over night, stimulated with 20% FBS (30 min), and lysed.
|Product Includes||Volume||Solution Color|
|Phospho-S6 Ribosomal Protein (Ser240/244) 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||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|
PathScan® Phospho-S6 Ribosomal Protein (Ser240/244) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of phosphorylated S6 ribosomal protein at Ser240/244. A Phospho-S6 Ribosomal Protein (Ser240/244) Rabbit mAb has been coated onto the microwells. After incubation with cell lysates, phospho-S6 ribosomal protein (Ser240/244) is captured by the coated antibody. Following extensive washing, an S6 Ribosomal Protein Mouse Detection mAb is added to detect the captured phospho-S6 ribosomal protein. 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 absorbance for the developed color is proportional to the quantity of S6 ribosomal protein phosphorylated at Ser240/244.
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
PathScan® Phospho-S6 Ribosomal Protein (Ser240/244) Sandwich ELISA Kit recognizes endogenous levels of phosphorylated S6 ribosomal protein in mouse and human cells, as shown in Figure 1. The kit sensitivity is shown in Figure 2. 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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