This Rhesus PD-1 overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of PD-1 protein (Cat: 90305-K08H) from the overexpression lysate was verified.
A DNA sequence encoding the rhesus PD1 (NP_001107830.1) (Met1-Gln167) was expressed with a polyhistidine tag at the C-terminus.
The recombinant rhesus PD1 consists 178 amino acids and predicts a molecular mass of 20.3 kDa.
Cell lysate was prepared by homogenization of the over-expressed cells in ice-cold modified RIPA Lysis Buffer with cocktail of protease inhibitors (Sigma). Cell debris was removed by centrifugation. Protein concentration was determined by Bradford assay (Bio-Rad protein assay, Microplate Standard assay). The cell lysate was boiled for 5 min in 1 x SDS loading buffer (50 mM Tris-HCl pH 6.8, 12.5% glycerol, 1% sodium dodecylsulfate, 0.01% bromophenol blue) containing 5% b-mercaptoethanol, and lyophilized.
Modified RIPA Lysis Buffer: 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% SDS, 1% Sodium deoxycholate, 1mM PMSF.
1. Centrifuge the tube for a few seconds and ensure the pellet at the bottom of the tube.
2. Re-dissolve the pellet using 200μL pure water and boil for 2-5 min.
1 X Sample Buffer (1 X modified RIPA buffer+1 X SDS loading buffer).
Stability & Storage
Store at 4℃ for up to twelve months from date of receipt. After re-dissolution, aliquot and store at -80℃ for up to twelve months. Avoid repeated freeze-thaw cycles.
Western Blot (WB)
Optimal dilutions/concentrations should be determined by the end user.
Programmed cell death 1, also known as PDCD1, is a type I transmembrane glycoprotein, and is an immunoreceptor belonging to the CD28/CTLA-4 family negatively regulates antigen receptor signaling by recruiting protein tyrosine phosphatase, SHP-2 upon interacting with either of two ligands, PD-L1 or PD-L2. PD1 inhibits the T-cell proliferation and production of related cytokines including IL-1, IL-4, IL-1 and IFN-γ by suppressing the activation and transduction of PI3K/AKT pathway. In addition, coligation of PD1 inhibits BCR-mediating signal by dephosphorylating key signal transducer. PD1 has been suggested to be involved in lymphocyte clonal selection and peripheral tolerance, and thus contributes to the prevention of autoimmune diseases. Furthermore, PD1 is shown to be a regulator of virus-specific CD8+ T cell survival in HIV infection. As a cell surface molecule, PDCD1 regulates the adaptive immune response. Engagement of PD-1 by its ligands PD-L1 or PD-L2 transduces a signal that inhibits T-cell proliferation, cytokine production, and cytolytic function.Immune CheckpointImmune Checkpoint Blockade: Blocking Antibodies Immune Checkpoint Blockade: PD1 / PDCD1 / CD279 Blocking AntibodiesImmune Checkpoint Detection: Antibodies Immune Checkpoint Detection: ELISA Antibodies Immune Checkpoint Detection: IHC Antibodies Immune Checkpoint Detection: WB AntibodiesImmune Checkpoint Proteins PD1 / PDCD1 / CD279 Immune Checkpoint ProteinsPD1 / PDCD1 / CD279 Immune Checkpoint PD1 / PDCD1 / CD279 Immune Checkpoint AntibodiesImmune Checkpoint Targets Co-inhibitory Immune Checkpoint Targets Immunotherapy Cancer Immunotherapy Targeted Therapy
James ES, et al. (2005) PDCD1: a tissue-specific susceptibility locus for inherited inflammatory disorders. Genes Immun. 6(5): 430-7.Okazaki T, et al. (2007) PD-1 and PD-1 ligands: from discovery to clinical application. Int Immunol. 19(7): 813-24.del Rio ML, et al. (2008) PD-1/PD-L1, PD-1/PD-L2, and other co-inhibitory signaling pathways in transplantation. Transpl Int. 21(11): 1015-28.Riley JL.(2009) PD-1 signaling in primary T cells. Immunol Rev. 229(1): 114-25.