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PE Mouse anti-CD22 (pY822)
PE Mouse anti-CD22 (pY822)

Analysis of CD22 (pY822) in human peripheral blood lymphocytes.  Human peripheral blood mononuclear cells (PBMC) were either stimulated (right panel) by cross-linking of surface IgM with purified Goat F(ab')2 Anti-Human IgM (SouthernBiotech) at 37°C for 2 minutes or unstimulated (left panel).  The cells were fixed with pre-warmed BD Phosflow™ Lyse/Fix Buffer (Cat. No. 558049) for 10 minutes at 37ºC, permeabilized with BD Phosflow™ Perm Buffer III (Cat. No. 558050) on ice for at least 30 minutes, and then stained with PE Mouse anti-CD22 (pY822, Cat. No. 560076) and Alexa Fluor® 647 Mouse anti-human CD20 (Cat. No. 558054).  For data analysis, lymphocytes were selected by their scatter profile.  The data demonstrates that the upregulated phosphorylation is restricted to the stimulated CD20-positive B lymphocytes.  Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.  We have also demonstrated that this antibody conjugate stains mouse CD22 (pY837) in splenocytes that are activated by IgM cross-linking (data not shown).

PE Mouse anti-CD22 (pY822)

The specificity of mAb 12a/CD22 was confirmed by western blot analysis using unconjugated antibody at 0.5 µg/ml on lysates from control (lane 1) and anti-IgM-stimulated (lane 2) mouse splenocytes.  Mouse CD22 (pY837) is identified as a band of ~140 kDa in the activated splenocytes.  Cross-reactivity to human CD22 (pY822) was demonstrated by western blot analysis on lysates from unstimulated and pervanadate-stimulated Daudi Burkitt's lymphoma cells (data not shown).

Analysis of CD22 (pY822) in human peripheral blood lymphocytes.  Human peripheral blood mononuclear cells (PBMC) were either stimulated (right panel) by cross-linking of surface IgM with purified Goat F(ab')2 Anti-Human IgM (SouthernBiotech) at 37°C for 2 minutes or unstimulated (left panel).  The cells were fixed with pre-warmed BD Phosflow™ Lyse/Fix Buffer (Cat. No. 558049) for 10 minutes at 37ºC, permeabilized with BD Phosflow™ Perm Buffer III (Cat. No. 558050) on ice for at least 30 minutes, and then stained with PE Mouse anti-CD22 (pY822, Cat. No. 560076) and Alexa Fluor® 647 Mouse anti-human CD20 (Cat. No. 558054).  For data analysis, lymphocytes were selected by their scatter profile.  The data demonstrates that the upregulated phosphorylation is restricted to the stimulated CD20-positive B lymphocytes.  Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.  We have also demonstrated that this antibody conjugate stains mouse CD22 (pY837) in splenocytes that are activated by IgM cross-linking (data not shown).

The specificity of mAb 12a/CD22 was confirmed by western blot analysis using unconjugated antibody at 0.5 µg/ml on lysates from control (lane 1) and anti-IgM-stimulated (lane 2) mouse splenocytes.  Mouse CD22 (pY837) is identified as a band of ~140 kDa in the activated splenocytes.  Cross-reactivity to human CD22 (pY822) was demonstrated by western blot analysis on lysates from unstimulated and pervanadate-stimulated Daudi Burkitt's lymphoma cells (data not shown).

Product Details
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BD Phosflow™
Human BL-CAM, Siglec-2, or Leu-14 (pY822); Mouse Lyb-8 (pY837)
Human, Mouse (QC Testing)
Mouse IgG1, κ
Phosphorylated Mouse CD22 Peptide
Intracellular staining (flow cytometry) (Routinely Tested)
20 µl
AB_10611569
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.

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. 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.
  4. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  5. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
560076 Rev. 2
Antibody Details
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12a/CD22

CD22 is a member of the immunoglobulin (Ig) superfamily that contains several extracellular Ig domains and cytoplasmic Immunoreceptor Tyrosine-based Inhibitory Motifs (ITIMs).  It is expressed in the cytoplasm of pro-B and pre-B cells and on the surface of IgD-positive mature B lymphocytes.  Differential splicing of the human CD22 gene results in the expression of two isoforms; the full-length CD22β has 7 Ig domains, while CD22α has only 5.  CD22β is the predominant isoform in human cells, at both the mRNA and protein levels.  Ligands for the extracellular domains of CD22 are sialic acid-linked glycoproteins on the B cell surface.  The intracellular ITIM motifs are substrates of src family tyrosine kinases.  Signal transduction is initiated by the phosphorylation of tyrosines in the ITIMs of CD22 upon ligation of the B cell receptor (BCR).  Tyrosine phosphorylation in ITIM3 of human CD22 or ITIM2 of mouse CD22 is required for recruitment of the protein tyrosine phosphatase PTP1C (SHP-1), which may terminate signal transduction by CD22 and by co-localized receptors, such as the BCR.  Studies of mice deficient for CD22 suggest that this adhesion molecule generates both negative and positive regulatory signals that affect B cell development and responsiveness.  

The 12a/CD22 monoclonal antibody recognizes the phosphorylated tyrosine 822 (pY822) in ITIM3 of human CD22β.  The equivalent site in human CD22α is pY645. The orthologous phosphorylation site in the predominant isoform of mouse CD22 is pY837, which is in ITIM2.

560076 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.
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PE
Yellow-Green 488 nm, 532 nm, 561 nm
496 nm, 566 nm
576 nm
560076 Rev.2
Citations & References
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Development References (3)

  1. Fujimoto M, Kuwano Y, Watanabe R, et al. B cell antigen receptor and CD40 differentially regulate CD22 tyrosine phosphorylation. J Immunol. 2006; 176:873-879. (Biology).
  2. Grewal :K, Boton M, Ramirez K, et al. ST6Gal-I restrains CD22-dependent antigen receptor endocytosis and Shp-1 recruitment in normal and pathogenic immune signaling. Mol Cell Biol. 2006; 26(13):4970-4981. (Biology).
  3. Tedder TF, Tuscano J, Sato S, Kehrl JH. CD22, a B lymphocyte-specific adhesion molecule that regulates antigen receptor signaling. Annu Rev Immunol. 1997; 15:481-504. (Biology).
560076 Rev. 2

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