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PE Mouse anti-S6 (pS235/pS236)
PE Mouse anti-S6 (pS235/pS236)

Analysis of S6 (pS235/pS236) in activated human peripheral blood mononuclear cells (PBMC). PBMC were isolated by density gradient centrifugation (Ficoll-Paque™ PLUS, Cat. No. 17-1440-02) and either left untreated (open histogram) or treated with PMA (Sigma-Aldrich, Cat. No. P8139) at 50 nM/10^6 cells for 30 minutes (shaded histogram). Cells were then fixed in BD Cytofix™ buffer (Cat. No. 554655) at 37°C for 10 minutes, then permeabilized with BD Phosflow™ Perm Buffer III (Cat. No. 558050) on ice for at least 30 minutes, and then stained with PE Mouse anti-S6 (pS235/pS236). For data analysis, lymphocytes were selected by scatter profile.  Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.

PE Mouse anti-S6 (pS235/pS236)

Western blot analysis of S6 (pS235/pS236).  The specificity of mAb N7-548 was confirmed by western blot analysis using unconjugated Mouse anti-S6 (pS235/pS236) antibody on lysates from untreated (lane 1) or PMA-treated (lane 2) PBMC. S6 (pS235/pS236) is identified as a band of 32 kDa, with increased intensity in the PMA-treated cells.  Purified Mouse anti-Actin monoclonal antibody (Cat. No. 612656 or 612657) was the gel-loading control.

Analysis of S6 (pS235/pS236) in activated human peripheral blood mononuclear cells (PBMC). PBMC were isolated by density gradient centrifugation (Ficoll-Paque™ PLUS, Cat. No. 17-1440-02) and either left untreated (open histogram) or treated with PMA (Sigma-Aldrich, Cat. No. P8139) at 50 nM/10^6 cells for 30 minutes (shaded histogram). Cells were then fixed in BD Cytofix™ buffer (Cat. No. 554655) at 37°C for 10 minutes, then permeabilized with BD Phosflow™ Perm Buffer III (Cat. No. 558050) on ice for at least 30 minutes, and then stained with PE Mouse anti-S6 (pS235/pS236). For data analysis, lymphocytes were selected by scatter profile.  Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.

Western blot analysis of S6 (pS235/pS236).  The specificity of mAb N7-548 was confirmed by western blot analysis using unconjugated Mouse anti-S6 (pS235/pS236) antibody on lysates from untreated (lane 1) or PMA-treated (lane 2) PBMC. S6 (pS235/pS236) is identified as a band of 32 kDa, with increased intensity in the PMA-treated cells.  Purified Mouse anti-Actin monoclonal antibody (Cat. No. 612656 or 612657) was the gel-loading control.

Product Details
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BD Phosflow™
40S ribosomal protein S6; Phosphoprotein NP33; RPS6; RS6
Human (QC Testing), Mouse, Rat (Predicted)
Mouse BALB/c IgG1, κ
Phosphorylated Human ribosomal protein S6 Peptide
Intracellular staining (flow cytometry) (Routinely Tested)
20 µl
AB_2827879
Aqueous buffered solution containing BSA and ≤0.09% sodium azide.
RUO


Preparation And Storage

Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated with R-PE under optimum conditions, and unconjugated antibody and free PE were removed.

Recommended Assay Procedures

This antibody conjugate is suitable for intracellular staining of human peripheral blood mononuclear cells using BD Cytofix™ Fixation Buffer.  Any of the three BD Phosflow™ permeabilization buffers may be used.

Product Notices

  1. This reagent has been pre-diluted for use at the recommended Volume per Test. We typically use 1 × 10^6 cells in a 100-µl experimental sample (a test).
  2. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
  3. Ficoll-Paque is a trademark of Amersham Biosciences Limited.
  4. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  5. All other brands are trademarks of their respective owners.
  6. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  7. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
560433 Rev. 3
Antibody Details
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N7-548

Ribosomal protein S6 (~29 kDa calculated and ~32 kDa observed molecular weights) is a component of the 40S ribosomal subunit and belongs to the S6E family of ribosomal proteins. The S6 ribosomal protein plays a role in regulating the translation of RNAs and thus controlling the growth and proliferation of cells. S6 ribosomal protein phosphorylation, especially at multiple C-terminal serine residues S235, S236, S240, and S244, activates S6. The activated S6 ribosomal protein in turn upregulates the ribosomal translation of RNA species coding for other ribosomal proteins, peptide elongation factors and other proteins involved in cell cycle entry and progression. These phosphorylations are mediated by various kinases (e.g., p70S6K and PKCD) activated through cellular responses to growth factors, cytokines, tumor promoting agents, and mitogens. The S6 ribosomal protein can be dephosphorylated in growth-arrested cells.

The N7-548 monoclonal antibody specifically detects the S6 ribosomal protein phosphorylated at S235 and S236.

560433 Rev. 3
Format Details
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PE
R-Phycoerythrin (PE), is part of the BD family of Phycobiliprotein dyes. This fluorochrome is a multimeric fluorescent phycobiliprotein with excitation maximum (Ex Max) of 496 nm and 566 nm and an emission maximum (Em Max) at 576 nm. PE is designed to be excited by the Blue (488 nm), Green (532 nm) and Yellow-Green (561 nm) lasers and detected using an optical filter centered near 575 nm (e.g., a 575/26-nm bandpass filter). As PE is excited by multiple lasers, this can result in cross-laser excitation and fluorescence spillover on instruments with various combinations of Blue, Green, and Yellow-Green lasers. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
altImg
PE
Yellow-Green 488 nm, 532 nm, 561 nm
496 nm, 566 nm
576 nm
560433 Rev.3
Citations & References
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Development References (9)

  1. Blatt K, Herrmann H, Mirkina I, et al. The PI3-kinase/mTOR-targeting drug NVP-BEZ235 inhibits growth and IgE-dependent activation of human mast cells and basophils. PLoS ONE. 2012; 7(1):e29925. (Clone-specific: Flow cytometry). View Reference
  2. Corradetti MN, Inoki K, Guan KL. The stress-inducted proteins RTP801 and RTP801L are negative regulators of the mammalian target of rapamycin pathway. J Biol Chem. 2005; 280(11):9769-9772. (Biology). View Reference
  3. Gu JJ, Santiago L, Mitchell BS. Synergy between imatinib and mycophenolic acid in inducing apoptosis in cell lines expressing Bcr-Abl. Blood. 2005; 105(8):3270-3277. (Biology). View Reference
  4. Jastrzebski K, Hannan KM, Tchoubrieva EB, Hannan RD, Pearson RB. Coordinate regulation of ribosome biogenesis and function by the ribosomal protein S6 kinase, a key mediator of mTOR function. Growth Factors. 2007; 25(4):209-226. (Biology). View Reference
  5. Kleijn M, Proud CG. The regulation of protein synthesis and translation factors by CD3 and CD28 in human primary T lymphocytes. BMC Biochem. 2002; 3:11. (Biology). View Reference
  6. Lal L, Li Y, Smith J, et al. Activation of the p70 S6 kinase by all-trans-retinoic acid in acute promyelocytic leukemia cells.. Blood. 2005; 105(4):1669-77. (Biology). View Reference
  7. Shah OJ, Ghosh S, Hunter T. Mitotic regulation of ribosomal S6 kinase 1 involves Ser/Thr, Pro phosphorylation of consensus and non-consensus sites by Cdc2. J Biol Chem. 2003; 278(18):16433-16442. (Biology). View Reference
  8. Shah OJ, Hunter T. Critical role of T-loop and H-motif phosphorylation in the regulation of S6 kinase 1 by the tuberous sclerosis complex. J Biol Chem. 2004; 279(20):20816-20823. (Biology). View Reference
  9. van de Laar L, van den Bosch A, Boonstra A, et al. PI3K-PKB hyperactivation augments human plasmacytoid dendritic cell development and function. Blood. 2012; 120(25):4982-4991. (Clone-specific: Flow cytometry). View Reference
View All (9) View Less
560433 Rev. 3

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For Research Use Only. Not for use in diagnostic or therapeutic procedures.