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Alexa Fluor® 488 Mouse anti-PDPK1 (pS241)
Alexa Fluor® 488 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 or untreated.  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.  LEFT PANEL:  The treated (solid line) and untreated (dashed line) PBMC were stained with Alexa Fluor® 488 Mouse IgG1, κ isotype control mAb MOPC-21 (Cat. No. 557782).  RIGHT PANEL:  The treated (solid line) and untreated (dashed line) PBMC were stained with Alexa Fluor® 488 Mouse anti-PDPK1 (pS241).  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 or untreated.  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.  LEFT PANEL:  The treated (solid line) and untreated (dashed line) PBMC were stained with Alexa Fluor® 488 Mouse IgG1, κ isotype control mAb MOPC-21 (Cat. No. 557782).  RIGHT PANEL:  The treated (solid line) and untreated (dashed line) PBMC were stained with Alexa Fluor® 488 Mouse anti-PDPK1 (pS241).  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_1645364
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 to Alexa Fluor® 488 under optimum conditions, and unreacted Alexa Fluor® 488 was 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. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
  3. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  4. Alexa Fluor® 488 fluorochrome emission is collected at the same instrument settings as for fluorescein isothiocyanate (FITC).
  5. The Alexa Fluor®, Pacific Blue™, and Cascade Blue® dye antibody conjugates in this product are sold under license from Molecular Probes, Inc. for research use only, excluding use in combination with microarrays, or as analyte specific reagents. The Alexa Fluor® dyes (except for Alexa Fluor® 430), Pacific Blue™ dye, and Cascade Blue® dye are covered by pending and issued patents.
  6. Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
  7. 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.
  8. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  9. An isotype control should be used at the same concentration as the antibody of interest.
560090 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.

560090 Rev. 2
Format Details
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Alexa Fluor™ 488
Alexa Fluor™ 488 Dye is part of the BD blue family of dyes. This is a small organic fluorochrome with an excitation maximum (Ex Max) at 494-nm and an emission maximum (Em Max) at 517-nm. Alexa Fluor™ 488 is designed to be excited by the Blue laser (488 nm) and detected using an optical filter centered near 520-nm (e.g., a 530/30-nm bandpass filter). Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
Alexa Fluor™ 488
Blue 488 nm
494 nm
517 nm
560090 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
560090 Rev. 2

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Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described


Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims.  Comparisons are not made against non-BD technologies, unless otherwise noted.

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