TIM-3 Overexpression Lysate

Western Blot: TIM-3 Overexpression Lysate (Adult Normal) [NBL1-11452] Left-Empty vector transfected control cell lysate (HEK293 cell lysate); Right -Over-expression Lysate for TIM-3.

• Reactivity: Hu
• Applications: WB

TIM-3 Overexpression Lysate Summary

• Description:

  TIM-3 Transient Overexpression Lysate

  
  Expression Host: HEK293T
  
  Plasmid: RC209440
  
  Accession#: NM_032782
  
  Protein Tag: C-MYC/DDK
  
  You will receive 1 vial of lysate (100ug), 1 vial of empty vector negative control (100ug), and 1 vial of 2xSDS sample buffer (250ul). Each vial of cell lysate contains 100ug of total protein (at 1 mg/ml). The 2xSDS Sample Buffer consists of 4% SDS, 125mM Tris-HCl pH6.8, 10% Glycerol, 0.002% Bromophenol blue, 100mM DTT.
• Gene: HAVCR2

Applications/Dilutions

• Dilutions:
  • Western Blot
• Application Notes: This product is intended for use as a positive control in Western Blot. Overexpression of the target protein was confirmed using an antibody to DDK (FLAG) epitope tag (NBP1-71705) present on the protein construct.
  
  Each vial of cell lysate contains 100ug of total protein which should be sufficient for 20-50 reactions. Depending on over-expression level, antibody affinity and detection system, some lysates can go as low as 0.1 ug per load. We recommend starting with 5ug of cell lysate. Add an equal amount of cell lysate and 2X SDS Sample buffer and boil the SDS samples for 10 minutes before loading.
• Theoretical MW: 33.2 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.

Packaging, Storage & Formulations

• Storage: Store at -80C. Avoid freeze-thaw cycles.
• Buffer: RIPA buffer

Lysate Details for Array

• Type: Overexpression

Notes

HEK293T cells in 10-cm dishes were transiently transfected with a non-lipid polymer transfection reagent specially designed and manufactured for large volume DNA transfection. Transfected cells were cultured for 48hrs before collection. The cells were lysed in modified RIPA buffer (25mM Tris-HCl pH7.6, 150mM NaCl, 1% NP-40, 1mM EDTA, 1xProteinase inhibitor cocktail mix, 1mM PMSF and 1mM Na3VO4, and then centrifuged to clarify the lysate. Protein concentration was measured by BCA protein assay kit.

Alternate Names for TIM-3 Overexpression 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