TIGIT Knockout HeLa Cell Lysate

Product Discontinued

TIGIT Knockout HeLa Cell Lysate Summary

• Preparation Method: Knockout achieved by using CRISPR/Cas9,-31 bp deletion in exon2 and Insertion of the selection cassette in exon2
• Gene: Tigit

Applications/Dilutions

• Dilutions:
  • Western Blot
• Application Notes: You will receive 1 vial (100ug) of knockout cell lysate and 1 vial (100ug) of Parental cell lysate. Lysate can be diluted with 1X SDS sample buffer and will be stable at -20 degrees C for 12 months. Minimize freeze-thaw cycles.

Packaging, Storage & Formulations

• Storage: Store at -20C short term. Aliquot and store at -80C long term. Avoid freeze-thaw cycles.
• Buffer: 0.1 mg cell homogenate lyophilized in RIPA buffer made with double-knockout cell lines.
• Concentration: LYOPH
• Reconstitution Instructions: To use as WB negative control, spin down briefly and resuspend in 100 uL 1xSDS sample buffer (2% SDS, 60 mM Tris-HCl pH 6.8, 10% Glycerol, 0.02% Bromophenol blue, 60 mM beta-mercaptoethanol). Boil the lysate for 3 - 5 minutes before loading it onto gel.

Lysate Details for Array

• Type: Knockout HeLa Cell

Notes

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Validation of antibody specificity is critical and verification of antibody performance against knockout samples is one way to guarantee that an antibody recognizes a specific target. Novus' KO cell lysate can be used as a negative control for western blots and to confirm the specificity of antibodies.

Alternate Names for TIGIT Knockout HeLa Cell Lysate

• T cell immunoreceptor with Ig and ITIM domains
• TIGIT
• VSIG9
• VSTM3
• WUCAM

Background

TIGIT (T cell Immunoreceptor with Ig and ITIM domains), also called Vstm3 (V-set and transmembrane domain-containing 3), Vsig9 (V-set and Ig domain-containing 9) and WUCAM (Washington University cell adhesion molecule), is a 30-34 kDa type I transmembrane protein that is a member of the CD28 family within the Ig superfamily of proteins (1-3). Human TIGIT cDNA encodes 244 amino acids (aa) including a 21 aa signal sequence, a 120 aa extracellular region with a V-type Ig-like domain and two potential N-glycosylation sites, a 21 aa transmembrane sequence, an 82 aa cytoplasmic domain with an immunoreceptor tyrosine-based inhibitory motif (ITIM) and has a theoretical molecular weight of ~27 kDa (3). TIGIT is expressed as a homodimer receptor on NK cells and subsets of activated, memory and regulatory T (Treg) cells, and on follicular helper T cells within secondary lymphoid organs (4). TIGIT has three known homodimer ligands that belong to the nectin family that are expressed on antigen presenting cells (APCs) or tumor cells (5). CD155 is the primary ligand for TIGIT, but it is also capable of binding CD112 and CD113 (5). The CD155/TIGIT signaling axis is an emerging immune checkpoint that inhibits function of T cells and NK cells. Upon TIGIT ligation, CD155 signaling leads to increased secretion of interleukin 10 (IL-10) and decreased secretion of proinflammatory cytokine IL-12 (5). TIGIT is a promising target for cancer immunotherapy and is the center of many pre-clinical studies investigating checkpoint blockade utilizing monoclonal antibodies either as a monotherapy or combination therapy (5).

TIGIT is commonly used as a marker for T cell exhaustion and its expression correlates with disease progression. Furthermore, in viral infections including HIV and SIV, TIGIT serves as a target for immune restoration (6). In cancer detection, TIGIT is upregulated and coexpressed with programmed cell death protein 1 (PD-1) on the majority of circulating tumor antigen (TA)-specific CD8+ T cells (7). In addition to this, TIGIT can inhibit immune cells at multiple steps within the cancer immunity cycle. These include inhibiting NK cell effector function, suppressing dendritic cell costimulatory abilities, suppressing CD8+ T cell effector function by Tregs or polio virus receptor (PVR)-stimulated myeloid cells, and by directly inhibiting CD8+ T cells and preventing elimination of cancer cells (7).

References

1. Joller, N., & Kuchroo, V. K. (2017). Tim-3, Lag-3, and TIGIT. Current topics in microbiology and immunology, 410, 127-156. https://doi.org/10.1007/82_2017_62

2. Xu, Z., Jin, B. A novel interface consisting of homologous immunoglobulin superfamily members with multiple functions. Cell Mol Immunol 7, 11-19 (2010). https://doi.org/10.1038/cmi.2009.108

3. Uniprot(P86176

4. Khan, M., Arooj, S., & Wang, H. (2020). NK Cell-Based Immune Checkpoint Inhibition. Frontiers in immunology, 11, 167. https://doi.org/10.3389/fimmu.2020.00167

5. Harjunpaa, H., & Guillerey, C. (2020). TIGIT as an emerging immune checkpoint. Clinical and experimental immunology, 200(2), 108-119. https://doi.org/10.1111/cei.13407

6. Blake, S. J., Dougall, W. C., Miles, J. J., Teng, M. W., & Smyth, M. J. (2016). Molecular Pathways: Targeting CD96 and TIGIT for Cancer Immunotherapy. Clinical cancer research: an official journal of the American Association for Cancer Research, 22(21), 5183-5188. https://doi.org/10.1158/1078-0432.CCR-16-0933

7. Manieri, N. A., Chiang, E. Y., & Grogan, J. L. (2017). TIGIT: A Key Inhibitor of the Cancer Immunity Cycle. Trends in immunology, 38(1), 20-28. https://doi.org/10.1016/j.it.2016.10.002

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Lysates are guaranteed for 6 months from date of receipt.

Bioinformatics

• Gene Symbol: Tigit