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Immunofluorescent analysis of Phospho-S6 pSer240/244 (green) in HeLa cells. Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA (Product # 37525) for 15 minutes at room temperature. Cells were probed without (left panel) or with (right panel) a Phospho-S6 pSer240/244 polyclonal antibody (Product # PA1-39503) at a dilution of 1:100 for at least 1 hour at room temperature, washed with PBS, and incubated with DyLight 488 goat anti-rabbit IgG secondary antibody (Product # 35552) at a dilution of 1:400 for 30 minutes at room temperature. F-Actin (red) was stained with DyLight 554 Phalloidin (Product # 21834) and nuclei (blue) were stained with Hoechst 33342 dye (Product # 62249). Images were taken on a Thermo Scientific ArrayScan or a ToxInsight Instrument at 20X magnification.
|Tested species reactivity||Human|
|Host / Isotype||Rabbit / IgG|
|Immunogen||Synthetic peptide derived from the region of human S6 ribosomal that contains phosphorylated serines 240 and 244|
|Purification||Antigen affinity chromatography|
|Storage buffer||PBS, pH 7.6, with 1% BSA|
|Contains||<0.1% sodium azide|
|Storage Conditions||4° C, do not freeze|
|Tested Applications||Dilution *|
|Immunofluorescence (IF)||1:100 - 1:500|
|Immunohistochemistry (Paraffin) (IHC (P))||1:50|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
Heat-mediated antigen retrieval is recommended prior to staining, using a 10mM citrate buffer, pH 6.0, for 10 minutes followed by cooling at room temperature for 20 min. Following antigen retrieval, incubate samples with primary antibody for 30 min at room temperature. A suggested positive control is breast carcinoma.
This antibody was originally validated as part of a Thermo Scientific Cellomics High Content Screening Kit. The antibody sold separately may have slightly different performance and may need to be further optimized for the best results.
40S ribosomal protein S6 (also known as RPS6) is a ~31 kDa substrate of p70 S6 kinase (p70S6K) and a major component of translational machinery involved in protein synthesis, cell growth, proliferation, and metabolism. RPS6 undergoes phosphorylation on multiple serines in the carboxyl terminal region in the order 236-->235-->240-->244-->247, due to the positions of these amino acid residues on the alpha-helix. Hyperphosphorylation of RPS6 stimulates protein synthesis that mediates progression through the cell cycle.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
K03E6.6; phosphoprotein NP33
OK/SW-cl.2; RPS6; S6