CD28 Antibody [Unconjugated]

Human CD28 Antigen Affinity-purified Polyclonal Antibody enhances IL-2 secretion in Jurkat human acute T cell leukemia cell line stimulated with 10 ng/mL phorbol myristate acetate (PMA) and 0.5 µM calcium ionophore, in a dose-dependent manner, as measured using the Quantikine Human IL-2 ELISA Kit (Catalog # D2050). The ED50 for this effect is typically 0.3-0.6 µg/mL.

CD28 was detected in immersion fixed human peripheral blood mononuclear cells (PBMCs) using 10 µg/mL Goat Anti-Human CD28 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-342-PB) for 3 hours at room temperature. Cells were stained with the NorthernLights™ 557-conjugated Anti-Goat IgG Secondary Antibody (red; Catalog # NL001) and counter-stained with DAPI (blue). View our protocol for Fluorescent ICC Staining of Non-adherent Cells.

CD28 was detected in immersion fixed paraffin-embedded sections of human tonsil using Goat Anti-Human CD28 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-342-PB) at 3 µg/mL for 1 hour at room temperature followed by incubation with the Anti-Goat IgG VisUCyte™ HRP Polymer Antibody (Catalog # VC004). Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue). Specific staining was localized to cell surfaces in lymphocytes. View our protocol for IHC Staining with VisUCyte HRP Polymer Detection Reagents.

• Reactivity: Hu
• Applications: Flow, IHC, CyTOF-ready, Func, ICC/IF
• Clonality: Polyclonal
• Host: Goat
• Conjugate: Unconjugated
• Concentration: LYOPH

CD28 Antibody [Unconjugated] Summary

• Immunogen: S. frugiperda insect ovarian cell line Sf 21-derived recombinant human CD28
  Asn19-Pro152
  Accession # P10747
• Specificity: Detects human CD28 in direct ELISAs and Western blots.
• Source: N/A
• Isotype: IgG
• Clonality: Polyclonal
• Host: Goat
• Gene: CD28
• Purity Statement: Antigen Affinity-purified
• Endotoxin Note: <0.10 EU per 1 μg of the antibody by the LAL method.

Applications/Dilutions

• Dilutions:
  • Agonist Activity 0.3-0.6 ug/mL
  • CyTOF-ready
  • Flow Cytometry 2.5 ug/10^6 cells
  • Immunocytochemistry 5-15 ug/mL
  • Immunohistochemistry 3-15 ug/mL

Packaging, Storage & Formulations

• 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.
• Buffer: Lyophilized from a 0.2 μm filtered solution in PBS with Trehalose. *Small pack size (SP) is supplied either lyophilized or as a 0.2 µm filtered solution in PBS.
• Preservative: No Preservative
• Concentration: LYOPH
• Reconstitution Instructions: Reconstitute at 0.2 mg/mL in sterile PBS.

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for CD28 Antibody [Unconjugated]

• CD28 antigen (Tp44)
• CD28 antigen
• CD28 molecule
• CD28
• MGC138290
• T-cell-specific surface glycoprotein CD28
• Tp44

Background

CD28 and CTLA-4, together with their ligands, B7-1 and B7-2, constitute one of the dominant costimulatory pathways that regulate T and B cell responses. CD28 and CTLA-4 are structurally homologous molecules that are members of the immunoglobulin (Ig) gene superfamily. Both CD28 and CTLA-4 are composed of a single Ig V‑like extracellular domain, a transmembrane domain and an intracellular domain. CD28 and CTLA-4 are both expressed on the cell surface as disulfide-linked homodimers or as monomers. The genes encoding these two molecules are closely linked on human chromosome 2 and mouse chromosome 1. Mouse CD28 is expressed constitutively on virtually 100% of mouse T cells and on developing thymocytes. Cell surface expression of mouse CD28 is down-regulated upon ligation of CD28 in the presence of PMA or PHA. In contrast, CTLA-4 is not expressed constitutively but is up-regulated rapidly following T cell activation and CD28 ligation. Cell surface expression of mouse CTLA-4 peaks approximately 48 hours after activation. Although both CTLA-4 and CD28 can bind to the same ligands, CTLA-4 binds to B7-1 and B7-2 with a 20-100 fold higher affinity than CD28. CD28/B7 interaction has been shown to prevent apoptosis of activated T cells via the upregulation of Bcl-x_L. CD28 ligation has also been shown to regulate Th1/Th2 differentiation.

1. Lenschow, D.J. et al. (1996) Annu. Rev. Immunol. 14:233.
2. Hathcock, K.S. and R.J. Hodes (1996) Advances in Immunol. 62:131.
3. Ward, S.G. (1996) Biochem. J. 318:361.

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

Publications for CD28 Antibody (AF-342-PB)(8)

Publications using AF-342-PB

• Popugailo, A;Rotfogel, Z;Levy, M;Turgeman, O;Hillman, D;Levy, R;Arad, G;Shpilka, T;Kaempfer, R; The homodimer interfaces of costimulatory receptors B7 and CD28 control their engagement and pro-inflammatory signaling Journal of biomedical science 2023-06-28 [PMID: 37381064] (Flow Cytometry, Mouse)
• H Yu, S Cui, Y Mei, Q Li, L Wu, S Duan, G Cai, H Zhu, B Fu, L Zhang, Z Feng, X Chen Mesangial Cells Exhibit Features of Antigen-Presenting Cells and Activate CD4+ T Cell Responses J Immunol Res, 2019-06-17;2019(0):2121849. 2019-06-17 [PMID: 31317046] (Cell Culture, Human)
• P Banerjee, M Ries, SK Janaka, AG Grandea, R Wiseman, DH O'Connor, TG Golos, DT Evans Diversification of Bw4 Specificity and Recognition of a Nonclassical MHC Class I Molecule Implicated in Maternal-Fetal Tolerance by Killer Cell Ig-like Receptors of the Rhesus Macaque J. Immunol., 2018-09-19;0(0):. 2018-09-19 [PMID: 30232137] (Flow Cytometry, Human)
• LB Boyette, C Macedo, K Hadi, BD Elinoff, JT Walters, B Ramaswami, G Chalasani, JM Taboas, FG Lakkis, DM Metes Phenotype, function, and differentiation potential of human monocyte subsets PLoS ONE, 2017-04-26;12(4):e0176460. 2017-04-26 [PMID: 28445506] (Functional Assay, Human)
• U Ellinghaus, A Cortini, CL Pinder, GL Friec, C Kemper, TJ Vyse Dysregulated CD46 shedding interferes with Th1-contraction in systemic lupus erythematosus Eur. J. Immunol., 2017-05-22;0(0):. 2017-05-22 [PMID: 28444759] (Functional Assay, Human)
• Gila Arad, Revital Levy, Iris Nasie, Dalia Hillman, Ziv Rotfogel, Uri Barash et al. Binding of Superantigen Toxins into the CD28 Homodimer Interface Is Essential for Induction of Cytokine Genes That Mediate Lethal Shock PLoS Biology 2011-09-13 [PMID: 21931534]
• Sakthivel P, Shively V, Kakoulidou M The soluble forms of CD28, CD86 and CTLA-4 constitute possible immunological markers in patients with abdominal aortic aneurysm. J. Intern. Med., 2007-04-01;261(4):399-407. 2007-04-01 [PMID: 17391115] (ELISA Development, Human)
• Bahlis NJ, King AM, Kolonias D, Carlson LM, Liu HY, Hussein MA, Terebelo HR, Byrne GE, Levine BL, Boise LH, Lee KP CD28-mediated regulation of multiple myeloma cell proliferation and survival. Blood, 2007-02-20;109(11):5002-10. 2007-02-20 [PMID: 17311991] (IHC, Human)

Bioinformatics

• Gene Symbol: CD28
• Uniprot:
  • P10747(Human)