Human PSGL-1/CD162 Antibody

  
  • Species Reactivity
    Human
  • Specificity
    Detects human PSGL-1/CD162 in direct ELISAs and Western blots.
  • Source
    Monoclonal Mouse IgG2A Clone # 688101
  • Purification
    Protein A or G purified from hybridoma culture supernatant
  • Immunogen
    Chinese hamster ovary cell line CHO-derived recombinant human PSGL-1/CD162
    Gln42-Gly295
    Accession # NP_002997
  • Formulation
    Lyophilized from a 0.2 μm filtered solution in PBS with Trehalose. *Small pack size (SP) is supplied as a 0.2 µm filtered solution in PBS.
  • Label
    Unconjugated
Applications
  •  
    Recommended
    Concentration
    Sample
  • Western Blot
    2 µg/mL
    See below
  • Flow Cytometry
    2.5 µg/106 cells
    See below
  • Immunohistochemistry
    5-25 µg/mL
    See below
  • CyTOF-ready
    Ready to be labeled using established conjugation methods. No BSA or other carrier proteins that could interfere with conjugation.
  • Immunocytochemistry
    8-25 µg/mL
    See below
Please Note: Optimal dilutions should be determined by each laboratory for each application. General Protocols are available in the Technical Information section on our website.
Data Examples
Detection of Human PSGL‑1/CD162 by Western Blot. Western blot shows lysates of Jurkat human acute T cell leukemia cell line. PVDF membrane was probed with 2 µg/mL of Mouse Anti-Human PSGL‑1/CD162 Monoclonal Antibody (Catalog # MAB9961) followed by HRP-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # HAF018). Specific bands weredetected for PSGL1/CD162 homodimer atapproximately 250 kDa and PSGL1/CD162monomer at approximately 110 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.
Detection of PSGL-1 in Human Peripheral Blood Monocytes by Flow Cytometry. Human peripheral blood monocytes were stained with Mouse Anti-Human PSGL‑1/CD162 Monoclonal Antibody (Catalog # MAB9961, filled histogram) or isotype control antibody (Catalog # MAB003, open histogram), followed by Allophycocyanin-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # F0101B).
Immunocytochemistry
PSGL‑1/CD162 in Human PBMCs.
PSGL‑1/CD162 was detected in immersion fixed human peripheral blood mononuclear cells (PBMCs) using Mouse Anti-Human PSGL‑1/CD162 Monoclonal Antibody (Catalog # MAB9961) at 8 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Mouse IgG Secondary Antibody (red; Catalog # NL007) and counterstained with DAPI (blue). Specific staining was localized to cytoplasm. View our protocol for Fluorescent ICC Staining of Non-adherent Cells.
Immunohistochemistry
PSGL‑1/CD162 in Human Tonsil.
PSGL‑1/CD162 was detected in immersion fixed paraffin-embedded sections of human tonsil using Mouse Anti-Human PSGL‑1/CD162 Monoclonal Antibody (Catalog # MAB9961) at 5 µg/mL for 1 hour at room temperature followed by incubation with the Anti-Mouse IgG VisUCyte™ HRP Polymer Antibody (Catalog # VC001). Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue). Specific staining was localized to cytoplasm. View our protocol for IHC Staining with VisUCyte HRP Polymer Detection Reagents.
Preparation and Storage
  • Reconstitution
    Sterile PBS to a final concentration of 0.5 mg/mL.
  • Shipping
    The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below. *Small pack size (SP) is shipped with polar packs. Upon receipt, store it immediately at -20 to -70 °C
  • Stability & Storage
    Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
    • 12 months from date of receipt, -20 to -70 °C as supplied.
    • 1 month, 2 to 8 °C under sterile conditions after reconstitution.
    • 6 months, -20 to -70 °C under sterile conditions after reconstitution.
Background: PSGL-1/CD162

Human PSGL-1 (P-Selectin Glycoprotein Ligand-1; also CD162), is a 120 kDa mucin-type glycoprotein that plays a key role in leukocyte adhesion (1-3). It is synthesized as a 412 amino acid (aa) preproprecursor that contains a 17 aa signal sequence, a 24 aa propeptide, a 279 aa extracellular domain (ECD), a 21 aa transmembrane segment and a 71 aa cytoplasmic region (4, 5). Following cleavage of the pre- and prosegments, it is expressed as a 240 kDa disulfide-linked homodimer. The extreme N-terminus (aa 1-16 of the mature molecule) contains one threonine (aa 16) and three tyrosines (aa 5, 7, and 10) that are involved in ligand binding. The Thr residue allows for O-linked glycosylation in the form of a core-2 structure (GalNAc-Gal) linked in a beta 1,6 bond to a sialylated Lewis X motif (GlcNAc linked to both Fuc and Gal with a terminal sialic acid residue) (1, 2, 5, 6, 7). The three tyrosine residues allow for sulfation (8, 9). When binding to P-selectin, Tyr sulfation and glycosylation are essential. Tyr7 provides the most efficient sulfate moiety, while Fuc and sialic acid are essentially mandatory (7). When binding to E-selectin, only carbohydrate is needed, while both carbohydrate and Tyr10 are used for L-selectin binding (6, 8). There are 16 decameric aa repeats in the ECD of the longform of PSGL-1. This form is referred to as the A allele, and represents 65 - 80% of the population. Alleles B and C show deletions of decameric repeats #2 (aa 132-141) plus #9 and 10 (aa 222-241), respectively. Shorter forms may show weaker binding to P-selectin (9, 10). Soluble forms of PSGL-1 are also known. Neutrophil elastase will cleave somewhere within repeats #5-9, while cathepsin G cleaves after Tyr7 (11). The loss of Tyr5 and 7 should impact binding affinity. PSGL-1 is found on virtually all leukocytes and macrophages/DC’s (1). Although there is similarity in the organization of the ECD between species, there is little aa identity. Human PSGL-1 ECD shares 51%, 52% and 43% aa sequence identity with equine, canine and mouse ECD, respectively.

  • References:
    1. Yang, J. et al. (1999) Thromb. Haemost. 81:1.
    2. Cummings, R.D. (1999) Braz. J. Med. Biol. Res. 32:519.
    3. McEver, R.P. and R.D. Cummings (1997) J. Clin. Invest. 100:485.
    4. Sako, D. et al. (1993) Cell 75:1179.
    5. Veldman, G.M. et al. (1995) J. Biol. Chem. 270:16470.
    6. Bernimoulin, M.P. et al. (2003) J. Biol. Chem. 278:37.
    7. Leppanen, A. et al. (2000) J. Biol. Chem. 275:39569.
    8. Sako, D. et al. (1995) Cell 83:323.
    9. Afshar-Kharghan, V. et al. (2001) Blood 97:3306.
    10. Lozano, M.L. et al. (2001) Br. J. Haematol. 115:969.
    11. Gardiner, E.E. et al. (2001) Blood 98:1440.
  • Long Name:
    P-Selectin Glycoprotein Ligand 1
  • Entrez Gene IDs:
    6404 (Human); 20345 (Mouse); 363930 (Rat)
  • Alternate Names:
    CD162 antigen; CD162; P-selectin glycoprotein ligand 1; PSGL1; PSGL-1; PSGL-1cutaneous lymphocyte-associated associated antigen; selectin P ligandCLA; SELPLG
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