Analysis of Pyk2 (pY402) in human peripheral blood lymphocytes. Human peripheral blood mononuclear cells (PBMC) were either untreated (left panel) or stimulated by cross-linking of CD3 and CD28 with NA/LE Mouse anti-Human CD3 mAb UCHT1 (Cat. No. 555329) and NA/LE Mouse anti-Human CD28 mAb CD28.2 (Cat. No. 555725) on ice for 15 minutes, followed by Purified Goat anti-Mouse Ig (Cat. No. 553998) on ice for 15 minutes, and then allowed to undergo phosphorylation at 37°C for 5 minutes (right panel). The cells were fixed (BD Cytofix™ buffer, Cat. No. 554655) for 10 minutes at 37°C, permeabilized (BD Phosflow™ Perm Buffer III, Cat. No. 558050) on ice for 30 minutes, blocked with normal mouse immunoglobulin, and then stained with Alexa Fluor® 647 Mouse anti-Pyk2 (pY402, Cat. No. 560256) and PE Mouse anti-human CD3 mAb SK7 (Cat. No. 347347). For data analysis, lymphocytes were selected by their scatter profile. The data demonstrates that the upregulated phosphorylation of Pyk2 was restricted to the CD3-positive T lymphocytes under these stimulation conditions. Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.
The specificity of mAb L68-1256.272 was confirmed by western blot analysis using unconjugated antibody on lysates from PBMC that were untreated (lane 1) or stimulated by cross-linking of CD3 and CD28 (lane 2). Pyk2 (pY402) is identified as a band of 116 kDa, with increased intensity in the stimulated cells.
Analysis of Pyk2 (pY402) in human peripheral blood lymphocytes. Human peripheral blood mononuclear cells (PBMC) were either untreated (left panel) or stimulated by cross-linking of CD3 and CD28 with NA/LE Mouse anti-Human CD3 mAb UCHT1 (Cat. No. 555329) and NA/LE Mouse anti-Human CD28 mAb CD28.2 (Cat. No. 555725) on ice for 15 minutes, followed by Purified Goat anti-Mouse Ig (Cat. No. 553998) on ice for 15 minutes, and then allowed to undergo phosphorylation at 37°C for 5 minutes (right panel). The cells were fixed (BD Cytofix™ buffer, Cat. No. 554655) for 10 minutes at 37°C, permeabilized (BD Phosflow™ Perm Buffer III, Cat. No. 558050) on ice for 30 minutes, blocked with normal mouse immunoglobulin, and then stained with Alexa Fluor® 647 Mouse anti-Pyk2 (pY402, Cat. No. 560256) and PE Mouse anti-human CD3 mAb SK7 (Cat. No. 347347). For data analysis, lymphocytes were selected by their scatter profile. The data demonstrates that the upregulated phosphorylation of Pyk2 was restricted to the CD3-positive T lymphocytes under these stimulation conditions. Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.
The specificity of mAb L68-1256.272 was confirmed by western blot analysis using unconjugated antibody on lysates from PBMC that were untreated (lane 1) or stimulated by cross-linking of CD3 and CD28 (lane 2). Pyk2 (pY402) is identified as a band of 116 kDa, with increased intensity in the stimulated cells.
Product Details
BD Phosflow™
Protein tyrosine kinase 2β, FAK2, FADK 2, CAKβ, CADTK, RAFTK
Human (QC Testing), Mouse,Rat (Predicted)
Mouse BALB/c IgG2b, κ
Phosphorylated Human Pyk2 Peptide
Intracellular staining (flow cytometry) (Routinely Tested)
20 µl
AB_1645438
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® 647 under optimum conditions, and unreacted Alexa Fluor® 647 was removed.
Recommended Assay Procedures
Either BD Cytofix™ fixation buffer or BD Phosflow™ Fix Buffer I may be used for cell fixation. Any of the three BD Phosflow™ permeabilization buffers may be used.
Product Notices
- 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).
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
- 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.
- Alexa Fluor® 647 fluorochrome emission is collected at the same instrument settings as for allophycocyanin (APC).
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- 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.
- Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
Companion Products
Stain Buffer (FBS) RUO
Size
500 mL
Cat No.
554656
Stain Buffer (BSA) RUO
Size
500 mL
Cat No.
554657
Perm Buffer III RUO
Size
125 mL
Cat No.
558050
Fixation Buffer RUO
Size
100 mL
Cat No.
554655
Fix Buffer I RUO
Size
250 mL
Cat No.
557870
Perm/Wash Buffer I RUO
Size
125 mL
Cat No.
557885
560256 Rev. 2
Antibody Details
L68-1256.272
Proline-rich tyrosine kinase 2 (Pyk2) is a member of the FAK subfamily of tyrosine protein kinases. Pyk2 responds to a wide variety of inflammatory and stress stimuli, such as cytokines, ligation of integrins or antigen receptors, mechanical strain, and cellular depolarization. A common characteristic of most Pyk2 stimuli is an increase in cytosolic free calcium. Pyk2 is involved in signaling pathways that regulate vital cellular functions in hematopoietic, epithelial, neuronal, endothelial, and other cell types. Activation of Pyk2 initiates its autophosphorylation at tyrosine 402 (Y402), followed by Src-mediated phosphorylation of other Pyk2 tyrosine sites, which enhances its activity and leads to downstream signal transduction through the MAPK and JNK pathways.
The L68-1256.272 monoclonal antibody recognizes the phosphorylated Y402 of activated Pyk2.
560256 Rev. 2
Format Details
Alexa Fluor™ 647
Alexa Fluor™ 647 Dye is part of the BD red family of dyes. This is a small organic fluorochrome with an excitation maximum (Ex Max) at 653-nm and an emission maximum (Em Max) at 669-nm. Alexa Fluor™ 647 is designed to be excited by the Red laser (627-640 nm) and detected using an optical filter centered near 670-nm (e.g., a 660/20 nm bandpass filter). Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
Alexa Fluor™ 647
Red 627-640 nm
653 nm
669 nm
560256 Rev.2
Citations & References
Development References (6)
-
Anand AR, Cucchiarini M, Terwilliger EF, Ganju RK. The tyrosine kinase Pyk2 mediates lipopolysaccharide-induced IL-8 expression in human endothelial cells. J Immunol. 2008; 180:5636-5644. (Biology).
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Avraham H, Park S-Y, Schinkmann K, Avraham S.. RAFTK/Pyk2-mediated cellular signalling. Cell Signal. 2000; 12(3):123-133. (Biology).
-
Faure C, Corvol J-C, Toutant M, et al. Calcineurin is essential for depolarization-induced nuclear translocation and tyrosine phosphorylation of PYK2 in neurons. J Cell Sci. 2007; 120:3034-3044. (Biology).
-
Gluck SL. Acid sensing in renal epithelial cells. J Clin Invest. 2004; 114(12):1696-1699. (Biology).
-
Orr AW, Murphy-Ullrich EM. Regulation of endothelial cell function by FAK and Pyk2. Front Biosci. 2004; 9:1254-1266. (Biology).
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Park, S-Y, Avraham HK, Avraham S. RAFTK/Pyk2 activation is mediated by trans-acting autophosphorylation in a Src-independent manner. J Biol Chem. 2004; 279(32):33315-33322. (Biology).
560256 Rev. 2
Please refer to Support Documents for Quality Certificates
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.
