Q: We recently purchased Recombinant Human TIM-3 Protein (NBP2-23118) and GITR/TNFRSF18 ( NBP2-52215-0.05mg). Can these be diluted with sterile Di water without harming the integrity/functionality of the proteins?

A: Recombinant Human TIM-3 Protein (NBP2-23118) and Recombinant Human GITR/TNFRSF18 Protein (NBP2-52215) have been tested in PAGE but we have not tested its bioactivity/functional aspects so far. The buffer for NBP2-23118 (20mM Tris-HCl buffer (pH 8.0) containing 0.4M urea, 10% glycerol) contains urea and glycerol, which indicates there is the solubility/aggregation issue at the high concentration. I anticipate that if diluting with sterile double distilled water, at the certain concentrations, the protein will not be stable and precipitated out. Unfortunately, our lab did not measure the thresholds for the precipitations. On the other hand, NBP2-52215 has phosphate buffered saline (pH 7.4) and 10% glycerol as the buffer, which maybe tolerable in the water dilution process; however we have not done so in the past.

Recombinant Human TIM-3 His Protein

Novus Biologicals | Catalog # NBP2-23118

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Product Specifications
Scientific Data
Preparation & Storage
Background
Product Documents
Specific Notices
Research Areas

Key Product Details

Source

E. coli

Tag

His

Applications

SDS-PAGE

View all TIM-3 Proteins and Enzymes »

Product Specifications

Description

A denatured recombinant protein with a N-Terminal His-tag and corresponding to the amino acids 22-202 of Human TIM-3

Source: E.coli

Amino Acid Sequence: MGSSHHHHHH SSGLVPRGSH MGSHMSEVEY RAEVGQNAYL PCFYTPAAPG NLVPVCWGKG ACPVFECGNV VLRTDERDVN YWTSRYWLNG DFRKGDVSLT IENVTLADSG IYCCRIQIPG IMNDEKFNLK LVIKPAKVTP APTLQRDFTA AFPRMLTTRG HGPAETQTLG SLPDINLTQI STLANELRDS RLANDLRDSG ATIRIG

Purity

>90%, by SDS-PAGE

Predicted Molecular Mass

22.7 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.

Application Notes

Denatured protein is most likely not the best option for functional studies. It is better suited for Western Blot (WB) or imaging assays.

Protein / Peptide Type

Recombinant Protein

Scientific Data Images for Recombinant Human TIM-3 His Protein

SDS-PAGE: Recombinant Human TIM-3 His Protein [NBP2-23118]

SDS-Page: Recombinant Human TIM-3 Protein [NBP2-23118] - 3ug by SDS-PAGE under reducing condition and visualized by coomassie blue stain

Formulation, Preparation, and Storage

NBP2-23118

Formulation	20 mM Tris-HCl buffer (pH 8.0), 0.4M UREA, 10% glycerol
Preservative	No Preservative
Concentration	1 mg/ml
Shipping	The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage	Store at 4C short term. Aliquot and store at -20C long term. Avoid freeze-thaw cycles.

Background: TIM-3

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

Long Name

T Cell Immunoglobulin Mucin-3

Alternate Names

CD366, HAVcr-2, HAVCR2, KIM-3, SPTCL, TIM3, TIMD3

Gene Symbol

HAVCR2

Additional TIM-3 Products

Product Documents for Recombinant Human TIM-3 His Protein

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Certificate of Analysis

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Product Specific Notices for Recombinant Human TIM-3 His Protein

This product is for research use only and is not approved for use in humans or in clinical diagnosis. This product is guaranteed for 1 year from date of receipt.

Related Research Areas

Acute Myeloid Leukemia

Cancer Stem Cell Markers

Co-stimulatory and Co-inhibitory Molecules

Infectious Diseases

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