Recombinant Anti-TIM 3 antibody [EPR22241] (ab241332)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [EPR22241] to TIM 3
- Suitable for: mIHC, WB, IP, IHC-P
- Reacts with: Mouse, Rat, Human
Related conjugates and formulations
Overview
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Product name
Anti-TIM 3 antibody [EPR22241]
See all TIM 3 primary antibodies -
Description
Rabbit monoclonal [EPR22241] to TIM 3 -
Host species
Rabbit -
Tested applications
Suitable for: mIHC, WB, IP, IHC-Pmore details
Unsuitable for: Flow Cyt or ICC/IF -
Species reactivity
Reacts with: Mouse, Rat, Human -
Immunogen
Recombinant fragment. This information is proprietary to Abcam and/or its suppliers.
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Positive control
- WB: Daudi and RAW 264.7 whole cell lysates (+/- treatment with PNGase F). IP: Daudi whole cell lysate. IHC-P: Human liver and lung cancer tissue; mouse spleen tissue; rat spleen tissue. mIHC: Human tonsil
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General notes
This product is a recombinant monoclonal antibody, which offers several advantages including:
- - High batch-to-batch consistency and reproducibility
- - Improved sensitivity and specificity
- - Long-term security of supply
- - Animal-free production
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.
Properties
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Form
Liquid -
Storage instructions
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle. -
Storage buffer
pH: 7.2
Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol, 0.05% BSA -
Concentration information loading...
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Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
EPR22241 -
Isotype
IgG -
Research areas
Associated products
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Alternative Versions
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Compatible Secondaries
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Immunohistochemistry reagents
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Isotype control
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab241332 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Application | Abreviews | Notes |
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mIHC |
Use at an assay dependent concentration.
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WB | (1) |
1/1000. Detects a band of approximately 50-60 kDa (predicted molecular weight: 33 kDa).
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IP |
1/30.
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IHC-P |
1/1000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
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Notes |
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mIHC
Use at an assay dependent concentration. |
WB
1/1000. Detects a band of approximately 50-60 kDa (predicted molecular weight: 33 kDa). |
IP
1/30. |
IHC-P
1/1000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. |
Target
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Function
Cell surface receptor implicated in modulating innate and adaptive immune responses. Generally accepted to have an inhibiting function. Reports on stimulating functions suggest that the activity may be influenced by the cellular context and/or the respective ligand (PubMed:24825777). Regulates macrophage activation (PubMed:11823861). Inhibits T-helper type 1 lymphocyte (Th1)-mediated auto- and alloimmune responses and promotes immunological tolerance (PubMed:14556005). In CD8+ cells attenuates TCR-induced signaling, specifically by blocking NF-kappaB and NFAT promoter activities resulting in the loss of IL-2 secretion. The function may implicate its association with LCK proposed to impair phosphorylation of TCR subunits, and/or LGALS9-dependent recruitment of PTPRC to the immunological synapse (PubMed:24337741, PubMed:26492563). In contrast, shown to activate TCR-induced signaling in T-cells probably implicating ZAP70, LCP2, LCK and FYN (By similarity). Expressed on Treg cells can inhibit Th17 cell responses (PubMed:24838857). Receptor for LGALS9 (PubMed:16286920, PubMed:24337741). Binding to LGALS9 is believed to result in suppression of T-cell responses; the resulting apoptosis of antigen-specific cells may implicate HAVCR2 phosphorylation and disruption of its association with BAG6. Binding to LGALS9 is proposed to be involved in innate immune response to intracellular pathogens. Expressed on Th1 cells interacts with LGALS9 expressed on Mycobacterium tuberculosis-infected macrophages to stimulate antibactericidal activity including IL-1 beta secretion and to restrict intracellular bacterial growth (By similarity). However, the function as receptor for LGALS9 has been challenged (PubMed:23555261). Also reported to enhance CD8+ T-cell responses to an acute infection such as by Listeria monocytogenes (By similarity). Receptor for phosphatidylserine (PtSer); PtSer-binding is calcium-dependent. May recognize PtSer on apoptotic cells leading to their phagocytosis. Mediates the engulfment of apoptotic cells by dendritic cells. Expressed on T-cells, promotes conjugation but not engulfment of apoptotic cells. Expressed on dendritic cells (DCs) positively regulates innate immune response and in synergy with Toll-like receptors promotes secretion of TNF-alpha. In tumor-imfiltrating DCs suppresses nucleic acid-mediated innate immune repsonse by interaction with HMGB1 and interfering with nucleic acid-sensing and trafficking of nucleid acids to endosomes (By similarity). Expressed on natural killer (NK) cells acts as a coreceptor to enhance IFN-gamma production in response to LGALS9 (PubMed:22323453). In contrast, shown to suppress NK cell-mediated cytotoxicity (PubMed:22383801). Negatively regulates NK cell function in LPS-induced endotoxic shock. -
Tissue specificity
Expressed in T-helper type 1 (Th1) lymphocytes. Expressed on regulatory T (Treg) cells after TCR stimulation. Expressed in dendritic cells and natural killer (NK) cells. Expressed in epithelial tissues. Expression is increased on CD4+ and CD8+ T-cells in chronic hepatitis C virus (HCV) infection. In progressive HIV-1 infection, expression is up-regulated on HIV-1-specific CD8 T-cells. -
Involvement in disease
May be involved in T-cell exhaustion associated with chronic viral infections such as with human immunodeficiency virus (HIV) and hepatitic C virus (HCV). -
Sequence similarities
Belongs to the immunoglobulin superfamily. TIM family.
Contains 1 Ig-like V-type (immunoglobulin-like) domain. -
Post-translational
modificationsO-glycosylated with core 1 or possibly core 8 glycans.
Phosphorylated on tyrosine residues; modestly increased after TCR/CD28 stimulation. Can be phosphorylated in the cytoplasmatic domain by FYN (By similarity). Phosphorylation at Tyr-265 is increased by stimulation with ligand LGALS9. -
Cellular localization
Membrane. Cell junction. Localizes to the immunological synapse between CD8+ T-cells and target cells. - Information by UniProt
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Database links
- Entrez Gene: 84868 Human
- Entrez Gene: 171285 Mouse
- Entrez Gene: 363578 Rat
- Omim: 606652 Human
- SwissProt: Q8TDQ0 Human
- SwissProt: Q8VIM0 Mouse
- SwissProt: P0C0K5 Rat
- Unigene: 710500 Human
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Alternative names
- CD366 antibody
- FLJ14428 antibody
- HAVcr-2 antibody
see all
Images
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This data was developed using ab242080, the same antibody clone in a different buffer formulation.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human tonsil labelling PD1 with ab243644 at 1/500 dilution (1.02 µg/mL) (D), CD8 with ab178089 at 1/100 dilution (0.83 μg/ml) (C) and TIM 3 with ab242080 at 1/500 dilution (2.09 μg/ml) (B). Opal Polymer HRP Ms + Rb was used as a secondary antibody, and DAPI was used for a nuclear counter stain. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.
Panel A: merged staining of anti-TIM 3 (green; Opal™690), anti-CD8 (red; Opal™520) and anti-PD1 (gray; Opal™570) on human tonsil.
Panel B: anti-TIM 3 stained on membrane of a subset of immune cells.
Panel C: anti-CD8 stained on membrane of a subset of T cells.
Panel D: anti-PD1 stained on membrane of a subset of lymphocytes.
The section was incubated in three rounds of staining: in the order of ab242080, ab243644 and ab178089 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.
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Immunohistochemical analysis of paraffin-embedded human lung cancer tissue labeling TIM 3 with ab241332 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Cytoplasmic and membranous staining on both infiltrated immunocytes and tumor cells of human lung cancer (PMID: 22586058) is observed. Counter stained with hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.
Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).
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Immunohistochemical analysis of paraffin-embedded mouse spleen tissue labeling TIM 3 with ab241332 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Cytoplasmic and membranous staining on subsets of immune cells of mouse spleen is observed. Counter stained with hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.
Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).
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Immunohistochemical analysis of paraffin-embedded human liver tissue labeling TIM 3 with ab241332 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Cytoplasmic and membranous staining on Kupffer cells of human liver (PMID: 27192565) is observed. Counter stained with hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.
Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).
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Immunohistochemical analysis of paraffin-embedded rat spleen tissue labeling TIM 3 with ab241332 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Cytoplasmic and membranous staining on subsets of immune cells of rat spleen is observed. Counter stained with hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.
Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).
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All lanes : Anti-TIM 3 antibody [EPR22241] (ab241332) at 1/1000 dilution
Lane 1 : Daudi (human Burkitt's lymphoma cell line) whole cell lysate at 20 µg
Lane 2 : Daudi whole cell lysate (treated with PNGase F) at 20 mg/ml
Lane 3 : RAW 264.7 (mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate at 20 µg
Lane 4 : RAW 264.7 whole cell lysate (treated with PNGase F) at 20 µg
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution
Developed using the ECL technique.
Predicted band size: 33 kDa
Observed band size: 40 kDa why is the actual band size different from the predicted?
Additional bands at: 50-60 kDa (possible glycosylated form)
Exposure time: 70 secondsBlocking and dilution buffer: 5% NFDM/TBST.
Lanes 3 & 4 were developed using a higher sensitivity ECL substrate.
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TIM 3 was immunoprecipitated from 0.35 mg of Daudi (human Burkitt's lymphoma cell line) whole cell lysate with ab241332 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab241332 at 1/500 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.
Lane 1: Daudi lysate 10 μg (Input).
Lane 2: ab241332 IP in Daudi whole cell lysate (+).
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab241332 in Daudi whole cell lysate (-).Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 3 minutes. -
Tissue Microarrays stained for "Anti-TIM 3 antibody [EPR22241]” using "ab241332" in immunohistochemical analysis. This table provides a detailed overview of positive (tick mark) and negative (cross mark) staining per sample type tested. The sections were pre-treated using Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). The sections were incubated with ab241332 at +4°C overnight followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer).
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Tissue Microarrays stained for "Anti-TIM 3 antibody [EPR22241]” using "ab241332" in immunohistochemical analysis. This table provides a detailed overview of positive (tick mark) and negative (cross mark) staining per sample type tested. The sections were pre-treated using Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). The sections were incubated with ab241332 at +4°C overnight followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer).
Protocols
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
Datasheets and documents
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SDS download
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Datasheet download
Certificate of Compliance
References (13)
ab241332 has been referenced in 13 publications.
- Liu XF et al. Physical activity improves outcomes of combined lenvatinib plus anti-PD-1 therapy in unresectable hepatocellular carcinoma: a retrospective study and mouse model. Exp Hematol Oncol 11:20 (2022). PubMed: 35379324
- Wang Y et al. Crosstalk Between Metabolism and Immune Activity Reveals Four Subtypes With Therapeutic Implications in Clear Cell Renal Cell Carcinoma. Front Immunol 13:861328 (2022). PubMed: 35479084
- Zhang Q et al. USP35 is a Potential Immunosuppressive Factor in Skin Cutaneous Melanoma. J Inflamm Res 15:3065-3082 (2022). PubMed: 35637872
- Yang J et al. Single-cell profiling reveals molecular basis of malignant phenotypes and tumor microenvironments in small bowel adenocarcinomas. Cell Discov 8:92 (2022). PubMed: 36104333
- Seliger B et al. Induction of pulmonary HLA-G expression by SARS-CoV-2 infection. Cell Mol Life Sci 79:582 (2022). PubMed: 36334153