Human TIGIT HEK293 Overexpression Lysate: Product Information
This Human TIGIT overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of TIGIT protein (Cat: 10917-H08H) from the overexpression lysate was verified.
A DNA sequence encoding the extracellular domain of human TIGIT (NP_776160.2) (Met 1-Phe 138) with a C-terminal polyhistidine tag was expressed.
The secreted recombinant human TIGIT comprises 128 amino acids with a predicted molecular mass of 14.2 kDa. As a result of glycosylation, human TIGIT migrates as an approximately 20.8 kDa band in SDS-PAGE under reducing conditions.
Human TIGIT HEK293 Overexpression Lysate: Usage Guide
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.
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.
Human TIGIT HEK293 Overexpression Lysate: Synonyms
Human VSIG9 Overexpression Lysate; Human VSTM3 Overexpression Lysate; Human WUCAM Overexpression Lysate
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. Also, 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.
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