TIM-3 Knockout HeLa Cell Lysate

Product Discontinued

TIM-3 Knockout HeLa Cell Lysate Summary

• Preparation Method: Knockout achieved by using CRISPR/Cas9
• Gene: HAVCR2

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 TIM-3 Knockout HeLa Cell Lysate

• CD366
• FLJ14428
• HAVCR2
• HAVcr-2
• hepatitis A virus cellular receptor 2
• kidney injury molecule-3
• KIM-3
• SPTCL
• T cell immunoglobulin mucin 3
• T cell immunoglobulin mucin-3
• T-cell immunoglobulin and mucin domain-containing protein 3
• TIM 3
• TIM3 T-cell membrane protein 3
• TIM3
• TIM-3
• TIMD3
• TIMD-3
• TIMD3KIM-3

Background

T cell immunoglobulin and mucin-domain containing 3 (TIM-3) is a type I transmembrane protein that functions in suppressing immune cell responses and is considered a checkpoint receptor (1,2). TIM-3 is an inhibitory receptor that was first identified as a cell marker for interferon-gamma (IFN-gamma)-producing CD4+ T helper (Th1) and CD8+ T cytotoxic (Tc1) cells (1,2). TIM-3 is also expressed on other immune cell types: regulatory T cells (Treg), natural killer (NK) cells, macrophages, and dendritic cells (DCs), as well as leukemia stem cells (LSCs) (1-3). Human TIM-3 protein is 301 amino acids (aa) in length with a theoretical molecular weight (MW) of 33.4 kDa and shares ~63% aa sequence identity with mouse TIM-3 (2,4). The TIM-3 protein has an extracellular IgV domain, a mucin and stalk domain with O- and N-glycosylation sites, a transmembrane domain, and an intracellular tail with conserved tyrosine residues (2,3). Ligands for TIM-3 include soluble galectin-9, high-mobility group protein B1 (HMGB1), phosphatidylserine (PtdSer), and carcinoembyronic antigen-related cell adhesion molecule-1 (CEACAM-1) (1-3,5). Each of these ligands interact with different regions of the TIM-3 IgV domain. In the absence of ligand binding, the unphosphorylated intracellular tyrosine residues of TIM-3 are associated with HLA-B associated transcript 3 (Bat3), which recruits Lck and this Bat3-Lck complex preserves T cell signaling (1-3,5). When TIM-3 is engaged, an intracellular signaling cascade is initiated to inhibit immune cell activation. (1-3,5). Specifically, the tyrosine residues of TIM-3 are phosphorylated, Bat3 is released, and this results in suppression of immune responses (1-3, 5). Persistent, sustained TIM-3 signaling eventually results in T cell exhaustion (1-3,5).

Dysregulation of TIM-3 expression is associated with autoimmune diseases as shown by studies where inhibition of TIM-3 using blocking antibodies worsened disease progression in experimental autoimmune encephalomyelitis (EAE) models of multiple sclerosis (MS) (2,3,5). Conversely, high levels of TIM-3 have been shown during viral infection as well as in many cancer types where its increased expression may be an indicator of poor prognosis (2,3,5). TIM-3 has emerged as a potential cancer immunotherapy target as preclinical studies blocking TIM-3 results in increased anti-tumor immunity and prevents tumor growth (3,5). Studies have suggested combination therapy of TIM-3 blockade with blockade of other checkpoint inhibitors such as programmed death 1 (PD-1) or lymphocyte activation gene 3 (LAG-3) is more effective than TIM-3 blockade alone (3,5).

References

1. Acharya N, Sabatos-Peyton C, Anderson AC. Tim-3 finds its place in the cancer immunotherapy landscape. J Immunother Cancer. 2020; 8(1):e000911. https://doi.org/10.1136/jitc-2020-000911

2. Das M, Zhu C, Kuchroo VK. Tim-3 and its role in regulating anti-tumor immunity. Immunol Rev. 2017; 276(1):97-111. https://doi.org/10.1111/imr.12520

3. Joller N, Kuchroo VK. Tim-3, Lag-3, and TIGIT. Curr Top Microbiol Immunol. 2017; 410:127-156. https://doi.org/10.1007/82_2017_62

4. Uniprot (Q8TDQ0)

5. Wolf Y, Anderson AC, Kuchroo VK. TIM3 comes of age as an inhibitory receptor. Nat Rev Immunol. 2020; 20(3):173-185. https://doi.org/10.1038/s41577-019-0224-6

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: HAVCR2