Anti-phospho-PD-1 (Tyr248) Antibody, clone 407.6G12

Programmed cell death protein 1 (UniProt: Q15116; also known as Protein PD-1, hPD-1, CD279) is encoded by the PDCD1 (also known as PD1) gene (Gene ID: 5133) in human. PD-1 is a monomeric inhibitory cell surface receptor involved in the regulation of T-cell function during immunity and tolerance. PD-1 and its ligands, PD-L1 and PD-L2 play a key role in the maintenance of peripheral tolerance, a process by which the quiescence of autoreactive mature T cells is maintained. PD-1 is a single-pass type I membrane protein, synthesized with a signal peptide (aa 1-20), which is subsequently cleaved off. The mature form contains an extracellular domain (aa 24-170), a transmembrane domain (aa 171-191), and a cytoplasmic domain (aa 192-288). PD-1 also contains a single N-terminal immunoglobulin variable region (IgV) like domain, an ITIM motif (aa 221-226) and an ITSM motif ( aa 247-251). Tyrosine 248 falls in ITSM motif and located within the intracellular domain of PD-1 and, phosphorylation of PD-1 at Tyrosine 248 is a significant post-translational modification that influences its function. Src family kinases, such as Lck and Fyn, are involved in the initial phosphorylation of PD-1. Upon binding of PD-1 to its ligands (PD-L1 or PD-L2), phosphorylation at Tyr248 triggers downstream signaling pathways. PD-1 phosphorylation at Tyr248 promotes the recruitment of the protein tyrosine phosphatase PTPN11/SHP-2 that mediates de-phosphorylation of key TCR proximal signaling molecules, such as ZAP70, PRKCQ/PKC theta and CD247/CD3zeta. The phosphorylation at Tyr248 is crucial for the full inhibitory effect of PD-1 on T-cell activity. It contributes to the suppression of T-cell proliferation, cytokine production, and cytotoxic activity. Studies demonstrate that phosphorylation of the ITSM motif of PD-1 marks dysfunctional T cells that may be rescued with PD-1 blockade. Detection of phospho-PD-1 in TILs is a potential biomarker for PD-1 immunotherapy responses. (Ref.: Bu, X., et al. (2021). Cancer Immunol Res. 9(12):1465-1475).

KLH-conjugated linear peptide corresponding to 13 amino acids surrounding phospho-tyrosine 248 from the C-terminal half of human PD-1 (CD279).

Quality Control Testing

Evaluated by Western Blotting in lysate from Jurkat cells treated with pervanadate (0.1 mM; 5 min.at 37C).

Western Blotting Analysis (WB): A 1:1000 dilution of this antibody detected PD-1 phosphorylated on tyrosine 248 in lysate from Jurkat cells treated with pervanadate (0.1 mM; 5 min. at 37C), but not in lysate from untreated Jurkat cells.

Tested Applications

Western Blotting Analysis: A representative lot detected PD-1 (CD279) in Western Blotting application (Bu, X., et al. (2021). Cancer Immunol Res. 9(12):1465-1475).

Flow Cytometry Analysis: A representative lot detected PD-1 (CD279) in Flow Cytometry application (Bu, X., et al. (2021). Cancer Immunol Res. 9(12):1465-1475).

Note: Actual optimal working dilutions must be determined by end user as specimens, and experimental conditions may vary with the end user.

Clone 407.6G12 is a mouse monoclonal antibody that specifically detects PD-1 (CD279) phosphorylated on tyrosine 248.

Purified mouse monoclonal antibody IgG2a in buffer containing 0.1 M Tris-Glycine (pH 7.4), 150 mM NaCl with 0.05% sodium azide.

1.0 mg/mL. Please refer to guidance on suggested starting dilutions and/or titers per application and sample type.

Recommended storage: +2°C to +8°C.

Concentration: Please refer to the Certificate of Analysis for the lot-specific concentration.

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