Human PSGL-1/CD162 Antibody Summary
Gln42-Gly295
Accession # Q14242
Applications
This antibody functions as an ELISA capture antibody when paired with Sheep Anti-Human PSGL‑1/CD162 Antigen Affinity-purified Polyclonal Antibody(Catalog # AF3345).
This product is intended for assay development on various assay platforms requiring antibody pairs. We recommend the Human PSGL-1/CD162 DuoSet ELISA (Catalog # DY3345-05) for convenient development of a sandwich ELISA.
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.
Scientific Data
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Detection of Human PSGL‑1/CD162 by Western Blot. Western blot shows lysates of human peripheral blood mononuclear cells (PBMCs). PVDF membrane was probed with 2 µg/mL of Mouse Anti-Human PSGL-1/CD162 Monoclonal Antibody (Catalog # MAB9962) followed by HRP-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # HAF018). A specific band was detected for PSGL-1/CD162 monomer at approximately 110-120 and PSGL-1/CD162 homodimer at approximately 250-260 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.
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Detection of PSGL-1/CD162 in Human Peripheral Blood Lymphocytes by Flow Cytometry. Human peripheral blood lymphocytes were stained with Mouse Anti-Human PSGL-1/CD162 Monoclonal Antibody (Catalog # MAB9962, filled histogram) or isotype control antibody (Catalog # MAB0041, open histogram) followed by anti-Mouse IgG PE-conjugates secondary antibody (Catalog # F0102B). View our protocol for Staining Membrane-associated Proteins.
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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 # MAB9962) at 5 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.
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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 # MAB9962) 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 lymphocytes. View our protocol for IHC Staining with VisUCyte HRP Polymer Detection Reagents.
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Human PSGL‑1/CD162 ELISA Standard Curve. Recombinant Human PSGL-1/CD162 protein was serially diluted 2-fold and captured by Mouse Anti-Human PSGL-1/CD162 Monoclonal Antibody (Catalog # MAB9962) coated on a Clear Polystyrene Microplate (Catalog # DY990). Sheep Anti-Human PSGL-1/CD162 Antigen Affinity-purified Polyclonal Antibody(Catalog # AF3345) was biotinylated and incubated with the protein captured on the plate. Detection of the standard curve was achieved by incubating Streptavidin-HRP (Catalog # DY998) followed by Substrate Solution (Catalog # DY999) and stopping the enzymatic reaction with Stop Solution (Catalog # DY994).
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Detection of Human PSGL‑1/CD162 by Western Blot. Western blot shows lysates of Jurkat human acute T cell leukemia cell line and Ramos human Burkitt's lymphoma cell line. PVDF membrane was probed with 2 µg/mL of Mouse Anti-Human PSGL‑1/CD162 Monoclonal Antibody (Catalog # MAB9962) followed by HRP-conjugated Anti-Mouse IgG Secondary Antibody (HAF018). Specific bands were detected for PSGL‑1/CD162 at approximately 120, 250 kDa (as indicated). This experiment was conducted under reducing conditions and using Western Blot Buffer Group 1.
Reconstitution Calculator
Preparation and Storage
- 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.
- Yang, J. et al. (1999) Thromb. Haemost. 81:1.
- Cummings, R.D. (1999) Braz. J. Med. Biol. Res. 32:519.
- McEver, R.P. and R.D. Cummings (1997) J. Clin. Invest. 100:485.
- Sako, D. et al. (1993) Cell 75:1179.
- Veldman, G.M. et al. (1995) J. Biol. Chem. 270:16470.
- Bernimoulin, M.P. et al. (2003) J. Biol. Chem. 278:37.
- Leppanen, A. et al. (2000) J. Biol. Chem. 275:39569.
- Sako, D. et al. (1995) Cell 83:323.
- Afshar-Kharghan, V. et al. (2001) Blood 97:3306.
- Lozano, M.L. et al. (2001) Br. J. Haematol. 115:969.
- Gardiner, E.E. et al. (2001) Blood 98:1440.
Product Datasheets
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