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Alexa Fluor® 647 Mouse anti-SHP2 (pY542)
Alexa Fluor® 647 Mouse anti-SHP2 (pY542)

Analysis of SHP2 (pY542) in activated human lymphocytes. Left panel: Peripheral blood mononuclear cells (PBMC) were either 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 1-2 minutes (shaded histogram) or unstimulated (open histogram). The PBMC were then fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655) for 10 minutes at 37ºC, permeabilized with BD Phosflow™ Perm Buffer III (Cat. No. 558050) on ice for at least 30 minutes, blocked with normal mouse immunoglobulin, and then stained with Alexa Fluor® 647 Mouse anti-SHP2 (pY542).  For data analysis, lymphocytes were selected by their scatter profile. Flow cytometry was performed on a BD FACSCanto™ II flow cytometry system.

The specificity of mAb L99-921 was confirmed by western blot analysis using unconjugated antibody on lysates from mouse and human cells.  Middle panel: Lysates from mouse NIH-3T3 cells that had been treated with 200 nM PDGF (Cat. No. 354051) for 30 minutes (right blot) or untreated (left blot) were probed with purified mouse anti-SHP2 (pY542) monoclonal antibody at concentrations of 1.0, 0.25 and 0.063 μg/ml (Lanes 1, 2, and 3, respectively).  SHP2 (pY542) is identified as a band of 65-72 kDa in the treated cells.  Right panel: Lysates from PBMCs that were untreated (lane 1) or stimulated by cross-linking of CD3 and CD28 (lane 2) were probed with 0.5 μg/ml purified mouse anti-SHP2 (pY542) monoclonal antibody.  SHP2 (pY542) is identified as a band of 65-72 kDa, with increased intensity in the stimulated cells.

Analysis of SHP2 (pY542) in activated human lymphocytes. Left panel: Peripheral blood mononuclear cells (PBMC) were either 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 1-2 minutes (shaded histogram) or unstimulated (open histogram). The PBMC were then fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655) for 10 minutes at 37ºC, permeabilized with BD Phosflow™ Perm Buffer III (Cat. No. 558050) on ice for at least 30 minutes, blocked with normal mouse immunoglobulin, and then stained with Alexa Fluor® 647 Mouse anti-SHP2 (pY542).  For data analysis, lymphocytes were selected by their scatter profile. Flow cytometry was performed on a BD FACSCanto™ II flow cytometry system.

The specificity of mAb L99-921 was confirmed by western blot analysis using unconjugated antibody on lysates from mouse and human cells.  Middle panel: Lysates from mouse NIH-3T3 cells that had been treated with 200 nM PDGF (Cat. No. 354051) for 30 minutes (right blot) or untreated (left blot) were probed with purified mouse anti-SHP2 (pY542) monoclonal antibody at concentrations of 1.0, 0.25 and 0.063 μg/ml (Lanes 1, 2, and 3, respectively).  SHP2 (pY542) is identified as a band of 65-72 kDa in the treated cells.  Right panel: Lysates from PBMCs that were untreated (lane 1) or stimulated by cross-linking of CD3 and CD28 (lane 2) were probed with 0.5 μg/ml purified mouse anti-SHP2 (pY542) monoclonal antibody.  SHP2 (pY542) is identified as a band of 65-72 kDa, with increased intensity in the stimulated cells.

商品详情
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BD Phosflow™
PTN11, PTP1D, PTP2C, PTPN11, SH-PTP2, SH-PTP3, SHP-2, SHPTP2
Human (QC Testing), Mouse (Tested in Development), Rat (Predicted)
Mouse BALB/c IgG1, κ
Phosphorylated Human SHP2 Peptide
Intracellular staining (flow cytometry) (Routinely Tested), Western blot (Tested During Development)
20 µl
AB_1645439
Aqueous buffered solution containing BSA and ≤0.09% sodium azide.
RUO


准备和存储

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.

推荐的实验流程

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

商品通知

  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. Alexa Fluor® 647 fluorochrome emission is collected at the same instrument settings as for allophycocyanin (APC).
  4. 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.
  5. Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
  6. Ficoll-Paque is a trademark of Amersham Biosciences Limited.
  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. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  10. Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
560390 Rev. 3
抗体详情
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L99-921

SHP2 is a member of the cytosolic class of protein-tyrosine phosphatases (PTPs). SHP2 reportedly contains two SH2 domains, both of which are N-terminal to the PTP catalytic domain. SH2 PTPs are believed to work in conjunction with protein-tyrosine kinases to maintain intracellular protein phosphotyrosine homeostasis and cell cycle progression. The expression of SHP2 has been reported to be highest in brain, heart, and kidney. SHP2 is phosphorylated at two C-terminal tyrosine residues: Y542 and Y580. Phosphorylation of Y542 creates an interaction in the N-terminal SH2 domain and appears to relieve basal inhibition of the PTP, while phosphorylation at Y580 creates an interaction at the C-terminal that stimulates PTP activity.

The L99-921 monoclonal antibody recognizes the phosphorylated Y542 of SHP2 (PTP2C isoform 2). The homologous phosphorylation site in the PTP2Ci splice variant (isoform 1) is Y546. The specificity of this antibody was validated by confirming, using western blot analysis, that RNA-mediated interference (RNAi) of the specific protein was able to down-regulate the expression of SHP2 (pY542).

560390 Rev. 3
格式详情
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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 520-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
560390 Rev.3
报价单和参考
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研发参考 (6)

  1. Lu W, Gong D, Bar-Sagi D, Cole PA. Site-specific incorporation of a phosphotyrosine mimetic reveals a role for tyrosine phosphorylation of SHP-2 in cell signaling. Mol Cell. 2001; 8:759-769. (Biology). 查看参考
  2. Ahmad S, Banville D, Zhao Z, Fischer EH, Shen SH. A widely expressed human protein-tyrosine phosphatase containing src homology 2 domains. Proc Natl Acad Sci U S A. 1993; 90(6):2197-2201. (Biology). 查看参考
  3. Bennett AM, Tang TL, Sugimoto S, Walsh CT, Neel BG. Protein-tyrosine-phosphatase SHPTP2 couples platelet-derived growth factor receptor beta to Ras. Proc Natl Acad Sci U S A. 1994; 91(15):7335-7339. (Biology). 查看参考
  4. Fornasa G, Groyer E, Clement M, et al. TCR stimulation drives cleavage and shedding of the ITIM receptor CD31. J Immunol. 2010; 184(10):5485-5492. (Clone-specific: Flow cytometry). 查看参考
  5. MacGillivray M, Herrera-Abreu MT, Chow CW et al. The protein tyrosine phosphatase SHP-2 regulates interleukin-1-induced ERK activation in fibroblasts. J Biol Chem. 2003; 278(29):27190-27198. (Biology). 查看参考
  6. Marie-Cardine A, Kirchgessner H, Bruyns E et al. SHP2-interacting transmembrane adaptor protein (SIT), a novel disulfide-linked dimer regulating human T cell activation. J Exp Med. 1999; 189(8):1181-1194. (Biology). 查看参考
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560390 Rev. 3

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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.