Cynomolgus TIGIT HEK293 Overexpression Lysate

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Cynomolgus TIGIT HEK293 Overexpression Lysate: Product Information

Product Description
This Cynomolgus TIGIT overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of TIGIT protein (Cat: 90890-C02H) from the overexpression lysate was verified.
Expression Host
HEK293 Cells
Species
Cynomolgus
Sequence Information
A DNA sequence encoding the cynomolgus TIGIT (XP_015300911.1) (Met1-Pro209) was expressed with the Fc region of human IgG1 at the C-terminus.
Molecule Mass
The recombinant cynomolgus TIGIT consists of 421 amino acids and predicts a molecular mass of 47.2 kDa.

Cynomolgus TIGIT HEK293 Overexpression Lysate: Usage Guide

Preparation Method
Cell lysate was prepared by homogenization 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.
Lysis Buffer
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.
Recommend Usage
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. 3. Store the lyophilized cell lysate at 4℃. After re-dissolution, recommend to aliquot it into smaller quantities and store at -80℃.
Sample Buffer
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.
Application
Western Blot (WB)
Optimal dilutions/concentrations should be determined by the end user.

TIGIT Background Information

TIGIT, also known as V-set and transmembrane domain-containing protein 3 (VSTM3) or V-set and immunoglobulin domain-containing protein 9 (VSIG9) is a new surface protein containing an immunoglobulin variable domain, a transmembrane domain and an immunoreceptor tyrosine-based inhibitory motif (ITIM). TIGIT is expressed on regulatory, memory, activated T cells and NK cells. It binds PVR with high affinity, and PVRL2 with lower affinity, but not PVRL3. Knockdown of TIGIT with siRNA in human memory T cells did not affect T cell responses, however, TIGIT inhibits NK cytotoxicity directly through its ITIM. TIGIT suppresses T cell activation by promoting the generation of mature immunoregulatory dendritic cells. The binding of PVR to TIGIT on human dendritic cells enhanced the production of IL-1 and diminished the production of IL-12p4. In addition, TIGIT counter inhibits the NK-mediated killing of tumor cells and protects normal cells from NK-mediated cytotoxicity thus providing an "alternative self" mechanism for MHC class I inhibition.
Full Name
T cell immunoreceptor with Ig and ITIM domains
References
  • Ota T., et al.,(2004), Complete sequencing and characterization of 21,243 full-length human cDNAs. Nat. Genet. 36:40-45.
  • Bechtel S., et al., (2007), The full-ORF clone resource of the German cDNA consortium.BMC Genomics 8:399-399.
  • Yu X., Harden K., et al.,(2009), The surface protein TIGIT suppresses T cell activation by promoting the generation of mature immunoregulatory dendritic cells. Nat. Immunol. 10:48-57.
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