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PE Mouse anti-PDPK1 (pS241)
PE Mouse anti-PDPK1 (pS241)

Analysis of PDPK1 (pS241) in human peripheral blood mononuclear cells (PBMC).  PBMC were either treated with calyculin A plus okadaic acid for 30 minutes at 37ºC (solid line histograms) or untreated (dashed line histograms).  The cells were fixed with pre-warmed BD Phosflow™ Lyse/Fix Buffer (Cat. No. 558049) for 15 minutes at 37ºC and permeabilized with BD Phosflow™ Perm Buffer III (Cat. No. 558050) on ice for 30 minutes.  The treated and untreated PBMC were stained with PE Mouse IgG1, κ isotype control mAb MOPC-21 (Cat. No. 559320, left panel) or PE Mouse anti-PDPK1 (pS241, Cat. No. 560092, right panel).  The data demonstrates that the phosphorylation of PDPK1 is constitutive in all PBMC and that the level of phosphorylation increases when phosphatase activity is inhibited by the treatment.  Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.

Analysis of PDPK1 (pS241) in human peripheral blood mononuclear cells (PBMC).  PBMC were either treated with calyculin A plus okadaic acid for 30 minutes at 37ºC (solid line histograms) or untreated (dashed line histograms).  The cells were fixed with pre-warmed BD Phosflow™ Lyse/Fix Buffer (Cat. No. 558049) for 15 minutes at 37ºC and permeabilized with BD Phosflow™ Perm Buffer III (Cat. No. 558050) on ice for 30 minutes.  The treated and untreated PBMC were stained with PE Mouse IgG1, κ isotype control mAb MOPC-21 (Cat. No. 559320, left panel) or PE Mouse anti-PDPK1 (pS241, Cat. No. 560092, right panel).  The data demonstrates that the phosphorylation of PDPK1 is constitutive in all PBMC and that the level of phosphorylation increases when phosphatase activity is inhibited by the treatment.  Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.

Product Details
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BD Phosflow™
PDK1 (pS241), PKB Kinase (pS241)
Human (QC Testing), Mouse, Rat (Predicted)
Mouse IgG1, κ
Phosphorylated Human PDPK1 Peptide
Intracellular staining (flow cytometry) (Routinely Tested)
20 µl
AB_1645523
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 mAb was characterized by flow cytometry (Flow) and western blot analysis (WB) using these model systems:

Method                Species                Cells                Treatment                                        Fixation                                Perm buffer        Result

Flow                        Human                Jurkat                Calyculin A + Okadaic Acid                Lyse/Fix or Cytofix                III                        Up-regulation

Flow                        Human                Jurkat                Calyculin A + Okadaic Acid                Lyse/Fix                                I or II                Unsatisfactory

Flow                        Human                PBMC                Calyculin A + Okadaic Acid                Lyse/Fix                                III                        Up-regulation

WB                        Human                Jurkat                Calyculin A + Okadaic Acid                                                                                63 kDa

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. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  3. An isotype control should be used at the same concentration as the antibody of interest.
  4. 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.
  5. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  6. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
560092 Rev. 2
Antibody Details
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J66-653.44.17

The serine/threonine kinase 3-Phosphoinositide-Dependent Protein Kinase-1 (PDPK1, also known as PDK1) contributes to the activation of many important kinases in the insulin and IGF-1 signaling pathways.  It acts downstream of phosphatidylinositol 3-kinase (PI3-kinase) to phosphorylate residues in the activation loops of many cellular kinases, including protein kinase B (PKB/Akt), PKC isoforms, p70 S6 kinase, and PDPK1 itself.  The autophosphorylation of PDPK1 at serine 241 (S241) has recently been suggested to play a role in the regulation of PDPK1.  It has been proposed that PDPK1 activity plays a key role in the regulation of various cellular events such as cell proliferation, differentiation, and apoptosis.

The J666-653.44.17 monoclonal antibody recognizes the phosphorylated S241 in the activation loop of human PDPK1.  The orthologous phosphorylation site in mouse and rat PDPK1 is S244.

560092 Rev. 2
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
560092 Rev.2
Citations & References
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Development References (2)

  1. Komander D, Kular G, Deak M, Alessi DR, van Aalten DM. Role of T-loop phosphorylation in PDK1 activation, stability, and substrate binding. J Biol Chem. 2005; 280(19):18797-18802. (Biology). View Reference
  2. Wick MJ, Ramos FJ, Chen H, et al. Mouse 3-phosphoinositide-dependent protein kinase-1 undergoes dimerization and trans-phosphorylation in the activation loop. J Biol Chem. 2003; 278(44):42913-42919. (Biology). View Reference
560092 Rev. 2

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