TTK Antibody, HRP conjugated
Description
Biological Role of TTK
TTK is a 97 kDa serine/threonine/tyrosine kinase encoded by the TTK gene (UniProt ID: P33981) . Key functions include:
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Regulation of mitotic spindle assembly and chromosome segregation
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Enhancement of Aurora kinase B (AURKB) activity at centromeres
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Involvement in cell proliferation, particularly in cancers like triple-negative breast cancer (TNBC)
TTK overexpression correlates with poor prognosis in TNBC, making it a therapeutic target . Its role in maintaining genomic stability during mitosis underscores its research importance .
Western Blot
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Protocol:
Immunohistochemistry
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Staining Protocol:
ELISA
Specificity
Performance Factors
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Buffer Compatibility:
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Stability: HRP activity diminishes over time; stabilizers like LifeXtend™ recommended for prolonged storage .
Cancer Studies
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TTK depletion in TNBC cell lines reduces viability and induces apoptosis .
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Overexpression confirmed via IHC in 80% of TNBC biopsies compared to non-TNBC subtypes .
Therapeutic Potential
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
* **Cell Proliferation:** TTK is believed to be associated with cell proliferation.
* **Mitotic Checkpoint Signaling:** TTK phosphorylates MAD1L1, promoting the activation of the mitotic checkpoint signaling pathway.
* **Chromosome Alignment:** TTK enhances AURKB activity (via direct CDCA8 phosphorylation) at the centromere, contributing to proper chromosome alignment during mitosis. This is crucial for the correct distribution of chromosomes during cell division, ensuring genetic stability.
* **Mitotic Checkpoint:** TTK is essential for the mitotic checkpoint, which ensures that all chromosomes are properly attached to the spindle microtubules before cell division proceeds. This checkpoint prevents the formation of daughter cells with incorrect chromosome numbers, preventing aneuploidy.
- Treatment of a xenograft model of a CTNNB1-mutant cell line with the TTK inhibitor NTRC 0066-0 resulted in complete inhibition of tumor growth. Mutations in CTNNB1 are frequently observed in endometrial cancer and hepatocellular carcinoma, both of which exhibit high TTK expression. This suggests that mutant CTNNB1 could be a prognostic biomarker for drug response, enabling the selection of patients who are most likely to benefit from TTK inhibition. PMID: 28751540
- Following lentiviral shRNA knockdown in several human liver cancer cell lines, it was demonstrated that TTK promotes cell growth and spreading, protects against senescence, and decreases autophagy. Additionally, in an experimental animal model, in vitro knockdown of TTK effectively suppressed the intrahepatic growth of human HCC xenografts. These findings highlight the crucial role of TTK in promoting liver cancer cell growth and its potential as a therapeutic target. PMID: 27618777
- The expression of TTK in gallbladder cancer (GBC) is lower than in normal tissues. However, higher levels of TTK expression in GBC are correlated with longer overall survival. These observations suggest that TTK may play a complex role in GBC, potentially contributing to a more favorable prognosis in certain cases. PMID: 28883705
- TTK has been identified as a favorable prognostic biomarker associated with improved survival in patients with triple-negative breast cancer. PMID: 27833085
- Mps1 promotes checkpoint activation through a sequential phosphorylation cascade involving Knl1, Bub1, and Mad1. This sequential multi-target phosphorylation process makes the checkpoint highly responsive to Mps1 and kinetochore-microtubule attachment, ensuring proper cell division control. PMID: 28072388
- Both monopolar spindle 1 (MPS1) and miR-21 depletion suppressed glioblastoma (GBM) cell proliferation. Conversely, ectopic expression of miR-21 rescued GBM cell growth from MPS1 inhibition. These findings suggest a potential interplay between MPS1 and miR-21 in regulating GBM cell proliferation. PMID: 25991676
- The TTK gene on 6q14.1 encodes a dual-specificity protein kinase (hMps1), a key spindle assembly checkpoint protein that regulates proper chromosomal alignment and segregation during mitosis. PMID: 28777004
- Data indicate that Thr/Tyr kinase (TTK)/monopolar spindle 1 kinase (Mps-1) is overexpressed in malignant mesothelioma (MM) and that its expression correlates with poor patient outcomes. This suggests that TTK might be a potential prognostic biomarker and therapeutic target for MM. PMID: 28759042
- Distinct elements within the Mps1 N-terminal extension and tetratricopeptide repeat domains govern localization to either the kinetochore or the centrosome. This suggests that Mps1 localization is tightly regulated and plays a critical role in its function during mitosis. PMID: 27339139
- Inhibition of the novel mitochondrial function of Mps1 is sufficient to kill tumor cells. This discovery suggests that targeting Mps1's mitochondrial function could be a promising strategy for cancer therapy. PMID: 27383047
- Studies have identified point mutations in the catalytic domain of MPS1 (C604Y and C604W) that result in enzymes retaining catalytic activity but exhibiting resistance to protein kinase inhibitors. These mutations were investigated by analyzing the crystal structures of recombinant wild-type and mutant MPS1 with various protein kinase inhibitors bound to the ATP-binding pocket of the catalytic domain. Understanding these resistance mechanisms is crucial for developing effective MPS1 inhibitors. PMID: 28726638
- Mps1 plays a critical role in preventing aneuploidy-induced cell death in pancreatic cancer. PMID: 28380042
- HLF-mediated miR-132 directly suppresses TTK expression, thereby exerting inhibitory effects on cancer cell proliferation, metastasis, and radioresistance. This finding highlights the potential of targeting the HLF-miR-132-TTK pathway for cancer treatment. PMID: 27522003
- Mps1 undergoes sumoylation, which plays a crucial role in regulating its functions during mitosis. PMID: 26675261
- Results suggest that inhibiting monopolar spindle 1 kinase (MPS1) could be a therapeutic strategy for targeting tetraploid cancer cells. PMID: 26637805
- Cetn3 inhibits Mps1 autophosphorylation at Thr-676, a known site of T-loop autoactivation, and interferes with Mps1-dependent phosphorylation of Cetn2. Overexpression of Cetn3 in cells attenuates the incorporation of Cetn2 into centrioles and centrosome reduplication, while depletion of Cetn3 leads to the generation of extra centrioles. These observations suggest that Cetn3 regulates centrosome duplication and centriole biogenesis through its interaction with Mps1. PMID: 26354417
- Knockdown of Cdkn3 stabilizes Mps1 at centrosomes. PMID: 26586430
- TTK contributes to hepatocellular carcinoma tumorigenesis by promoting cell proliferation and migration. PMID: 26418879
- Interaction of the central domain of ARHGEF17 with Mps1 has been observed. PMID: 26953350
- Data show that TTK protein kinase (hMps1) interacts with proto-oncogene protein MDM2 both in vivo and in vitro. PMID: 26531827
- Depletion of Mps1 reduces tumor cell viability compared to normal cells. This finding suggests that Mps1 is essential for tumor cell survival and could be a promising target for cancer therapy. PMID: 26398286
- Mps1 plays a crucial role in chromosome alignment by orchestrating Ndc80C-MT interactions, ensuring proper chromosome segregation during mitosis. PMID: 26240331
- High Mps1 expression, at both mRNA and protein levels, has been associated with high tumor grade, high Ki67 expression, and worse survival, particularly in Triple Negative Breast Cancer. This suggests that Mps1 could be a valuable prognostic marker and potential therapeutic target for this aggressive form of breast cancer. PMID: 25731686
- Five point mutations in the kinase domain of mitotic checkpoint kinase MPS1 have been identified, conferring resistance against multiple inhibitors. This discovery highlights the need for developing novel MPS1 inhibitors that can overcome these resistance mechanisms. PMID: 26202014
- The N-terminal of Mps1 exhibits high sequence similarity to the classic NES (Nuclear Export Signal). A fusion of this motif with EGFP results in the dramatic exclusion of the fusion protein from the nucleus. This suggests that the N-terminal region of Mps1 plays a role in its nuclear localization and export. PMID: 25886724
- TTK was up-regulated in HCC specimens. Overexpression of TTK promoted cell proliferation, anchor-dependent colony formation, and resistance to sorafenib in HCC cells. These findings suggest that TTK plays a critical role in HCC development and progression and may contribute to drug resistance. PMID: 24905462
- The dual-specificity protein kinase TTK, a key mitotic checkpoint regulator with links to p53 signaling, has been shown to be a promising overall prognostic marker for Hepatocellular carcinoma in a large patient cohort. This further strengthens the importance of TTK as a potential target for HCC treatment and management. PMID: 24859455
- The checkpoint protein kinase monopolar spindle 1 (Mps1) directly bound to Ndc80C through two independent interactions. PMID: 26068854
- The amino-terminal localization module of the spindle assembly checkpoint protein kinase MPS1 directly interacts with the HEC1 (highly expressed in cancer 1) calponin homology domain in the NDC80 (nuclear division cycle 80) kinetochore complex in vitro, in a phosphorylation-dependent manner. This interaction suggests a crucial role for MPS1 in regulating kinetochore function and chromosome segregation. PMID: 26068855
- Results emphasize the significance of dynamic autophosphorylation of Mps1 in regulating accurate chromosome segregation and ensuring proper mitotic progression. PMID: 25265012
- Data suggest that MPS1 kinase inhibition could be a viable treatment strategy for pancreatic ductal adenocarcinoma (PDAC). PMID: 24282275
- High TTK protein expression has been associated with pancreatic cancer. PMID: 25137017
- PP2A-B56 is a key phosphatase for the removal of the Mps1-mediated Knl1 phosphorylations, which are necessary for Bub1/BubR1 recruitment in mammalian cells. PMID: 25246613
- Findings suggest that high levels of Mps1 contribute to tumorigenesis by attenuating the spindle assembly checkpoint. PMID: 25063032
- Results provide evidence of a newly identified hMps1 phosphorylation site involved in the mitotic checkpoint, and that CHK2 contributes to chromosomal stability through hMps1. PMID: 24764296
- Mps1 governs chromosomal organization during the early stage of mitosis to facilitate proper chromosome segregation. PMID: 24934155
- MPS1 inhibitors may exert robust anticancer activity, either as standalone therapeutic interventions or combined with microtubule-targeting chemicals. This highlights the potential of MPS1 inhibitors as a promising therapeutic approach for cancer treatment. PMID: 23933817
- Data show that TTK protein kinase, lymphocyte antigen 6 complex locus K, and insulin-like growth factor (IGF)-II mRNA binding protein 3 are tumor-associated antigens recognized by cytotoxic T lymphocytes and HLA-A24-restricted epitope peptides. PMID: 17784873
- Sustained MPS1 activity is required for maintaining both the MAD1.C-MAD2 complex and open MAD2 (O-MAD2) at unattached kinetochores to facilitate C-MAD2 production. PMID: 24151075
- A novel role for Aurora B-Hec1-Mps1 signaling axis in governing accurate chromosome segregation in mitosis has been identified. PMID: 24187132
- Two proteins that interact with BLM, RMI1 and RMI2, are phosphorylated upon SAC activation, and, like BLM, RMI1, and RMI2, are phosphorylated in an MPS1-dependent manner. PMID: 24108125
- MPS1 is a protein kinase that is overexpressed in triple-negative breast cancer. PMID: 23700430
- Mps1 is an acidophilic kinase with a striking tendency for phosphorylation of threonines. PMID: 23510141
- Ultraviolet-C irradiation delays mitotic progression by recruiting Mps1 to kinetochores. PMID: 23531678
- Data propose that Chk1 and Mps1 jointly regulate Aurora-B, MCAK, Kif2b, and Hec1 to correct merotelic attachments. These findings suggest a role for Chk1 and Mps1 in error correction during cell division. PMID: 23321637
- Oncogenic B-Raf(V600E) abrogates the AKT/B-Raf/Mps1 interaction in melanoma cells. PMID: 23726842
- A VDAC3-Mps1 module at the centrosome promotes ciliary disassembly during cell cycle entry. PMID: 23388454
- Mps1 stimulates Aurora B recruitment to the centromere. PMID: 22732840
- These data are consistent with a model in which Aurora B activity relieves a tetratricopeptide repeat domain-dependent inhibitory constraint on MPS1 localization. PMID: 23569217
- It is proposed that persistent phosphorylation of Mps1 through BRAF(V600E) signaling is a key event in disrupting the control of centrosome duplication and chromosome stability, potentially contributing to tumorigenesis. PMID: 22430208
Q&A
What is TTK protein and what are its primary cellular functions?
TTK (also known as Mps1) is a dual-specificity protein kinase that phosphorylates proteins on serine, threonine, and tyrosine residues. It is primarily associated with cell proliferation and plays essential roles in chromosome alignment and the mitotic checkpoint. Specifically, TTK enhances Aurora B kinase (AURKB) activity at the centromere through direct phosphorylation of CDCA8, which is crucial for proper chromosome alignment during mitosis. This activity is essential for maintaining genomic stability during cell division .
What applications are TTK antibodies, particularly HRP-conjugated versions, suitable for?
TTK antibodies, especially HRP-conjugated versions, are utilized in several key applications in academic research:
| Application | Typical Dilution Range | Notes |
|---|---|---|
| Western Blot (WB) | 1:300-5000 | Detection of TTK protein in cell/tissue lysates |
| ELISA | 1:500-10000 | Quantitative detection of TTK |
| IHC-P | 1:100-400 | Detection in paraffin-embedded tissues |
| IHC-F | 1:100-500 | Detection in frozen tissue sections |
HRP conjugation provides signal amplification through enzymatic reaction with substrate, enabling sensitive detection without the need for secondary antibody incubation .
What are the recommended storage conditions for TTK antibody, HRP conjugated?
For optimal preservation of activity, TTK antibody, HRP conjugated should be stored at -20°C for long-term storage. For frequent use, 4°C storage for up to one month is acceptable. It is crucial to avoid repeated freeze-thaw cycles as they can significantly compromise antibody activity and HRP enzyme function. Commercial formulations typically contain 50% glycerol, 0.5-1% BSA, and buffer components that maintain stability. Importantly, sodium azide should be avoided in HRP-conjugated antibodies as it irreversibly inhibits HRP activity .
What species reactivity can be expected with commercially available TTK antibodies?
Commercial TTK antibodies demonstrate varying species reactivity profiles:
| Product Source | Reactive Species |
|---|---|
| Bioss | Human, Mouse, Rat, Dog, Cow, Pig, Rabbit |
| Boster Bio | Human, Mouse |
| Cusabio | Human |
When working with species not explicitly validated by manufacturers, researchers should perform thorough validation experiments before proceeding with full-scale studies .
How does TTK regulation influence mitotic checkpoint function and cell cycle progression?
TTK plays a critical role in the spindle assembly checkpoint (SAC), which prevents anaphase onset until all chromosomes are properly attached to the mitotic spindle. Its kinase activity directly impacts chromosome alignment by enhancing AURKB activity through phosphorylation of CDCA8 at the centromere. This phosphorylation cascade is essential for monitoring proper chromosome alignment and preventing chromosomal instability .
When investigating TTK's role in mitotic progression, researchers should consider:
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Cell synchronization methods to enrich for mitotic populations
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Co-staining with mitotic markers (phospho-histone H3, cyclin B1)
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Using TTK inhibitors as experimental controls
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Correlation of TTK phosphorylation status with mitotic checkpoint activation
What methodological considerations are important when using TTK antibody, HRP conjugated in Western blot applications?
For optimal Western blot performance with TTK antibody, HRP conjugated:
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Sample preparation:
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Include protease and phosphatase inhibitors
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Use appropriate lysis buffers (RIPA or NP-40 based)
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Clear lysates by high-speed centrifugation
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Buffer optimization:
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Blocking buffer: 5% non-fat dry milk or 3-5% BSA in TBST
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Antibody diluent: 1% BSA in TBST (ensure absence of sodium azide)
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Wash buffer: TBST with thorough washing (4-5 times for 5-10 minutes each)
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Antibody concentration:
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Start with 1:500-1:2000 dilution
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Optimize based on signal-to-noise ratio
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Detection considerations:
How can specificity of TTK antibody be rigorously validated in research applications?
Comprehensive validation of TTK antibody specificity should include:
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Positive and negative controls:
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Cell lines with known TTK expression levels
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Tissue panels with documented TTK expression patterns
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Genetic manipulation:
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siRNA/shRNA knockdown of TTK
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CRISPR/Cas9 knockout validation
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Overexpression systems
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Biochemical validation:
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Peptide competition assay with immunizing peptide
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Immunoprecipitation followed by mass spectrometry
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Comparison with multiple antibodies targeting different TTK epitopes
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Application-specific controls:
What approaches are recommended for optimizing fixation and antigen retrieval when using TTK antibody in immunohistochemistry?
Fixation and antigen retrieval significantly impact TTK antibody performance in immunohistochemistry:
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Fixation recommendations:
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10% neutral-buffered formalin (24-48 hours optimal)
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Avoid overfixation which can mask epitopes
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Alternative fixatives (methanol/acetone) may preserve certain epitopes better
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Antigen retrieval optimization:
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Heat-induced epitope retrieval (HIER)
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Trial both citrate buffer (pH 6.0) and EDTA buffer (pH 9.0)
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Optimize retrieval time (typically 10-30 minutes)
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Blocking considerations:
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Use serum from the same species as the secondary antibody
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Consider specialized blocking for endogenous peroxidase and biotin
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Dilution recommendation:
What are the optimal buffer conditions for antibody conjugation when preparing custom TTK-HRP conjugates?
When preparing custom TTK-HRP conjugates, buffer conditions are critical for conjugation efficiency:
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Recommended buffer composition:
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10-50mM amine-free buffer (HEPES, MES, MOPS, phosphate)
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pH range: 6.5-8.5
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Avoid buffers containing nucleophilic components (primary amines, thiols)
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Tris buffer can be tolerated at low concentrations (<20mM)
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Components to avoid:
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Sodium azide (irreversible HRP inhibitor)
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Thiomersal/thimerosal (contains reactive thiols)
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Primary amine-containing buffers
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Optimal antibody conditions:
What strategies can reduce background signal when using TTK antibody, HRP conjugated in immunoassays?
To optimize signal-to-noise ratio with TTK antibody, HRP conjugated:
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Blocking optimization:
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Test different blocking agents (5% milk, 3-5% BSA, commercial blockers)
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Increase blocking time (1-2 hours at room temperature or overnight at 4°C)
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Antibody dilution optimization:
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Start with manufacturer's recommendation
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Prepare a dilution series to identify optimal concentration
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For Western blot: typically 1:500-1:2000
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For IHC: typically 1:100-1:300
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For ELISA: typically 1:10000
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Washing protocol enhancement:
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Increase number of washes (4-5 times)
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Extend wash duration (5-10 minutes per wash)
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Consider adding 0.1-0.2% Tween-20 to reduce non-specific binding
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HRP-specific considerations:
What controls should be implemented when studying TTK expression across different cell cycle phases?
For rigorous cell cycle analysis of TTK expression:
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Cell synchronization controls:
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Synchronized populations at defined cell cycle phases
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Nocodazole or thymidine block protocols for mitotic enrichment
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Serum starvation for G0/G1 arrest
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Expression validation controls:
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Cell lines with characterized TTK expression patterns
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Genetic manipulation (knockdown/knockout/overexpression)
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Pharmacological TTK inhibitor controls (e.g., reversine)
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Co-staining validation:
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Cell cycle phase markers (cyclin proteins, Ki-67, phospho-histone H3)
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Co-localization with centromere/kinetochore markers
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Nuclear counterstaining (DAPI, Hoechst)
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Quantification approaches:
How can cross-reactivity issues be addressed when using TTK antibody in cross-species studies?
When applying TTK antibody across multiple species:
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Epitope analysis approach:
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Compare sequence homology of the immunogen region across target species
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Focus on antibodies raised against conserved regions when multi-species detection is required
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Review available reactivity data from manufacturers
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Experimental validation strategy:
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Test antibody performance on known positive controls from each species
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Include negative controls (knockdown/knockout samples if available)
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Perform peptide competition assays with species-specific peptides
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Application optimization:
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Adjust antibody concentration for each species separately
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Modify buffer conditions based on species-specific requirements
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Consider species-specific secondary antibodies for indirect detection methods
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Alternative approaches when cross-reactivity fails:
What quantification methods are most appropriate for measuring TTK expression levels across different applications?
Optimal quantification methods vary by application:
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Western blot quantification:
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Densitometry with normalization to loading controls (β-actin, GAPDH)
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Establish linear range using dilution series
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Use digital imaging systems with appropriate dynamic range
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Immunohistochemistry quantification:
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H-score system (intensity × percentage positive cells)
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Digital image analysis for staining intensity
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Comparison with known positive/negative controls
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Immunofluorescence quantification:
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Integrated density measurements
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Mean fluorescence intensity
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Colocalization analysis with cell cycle markers
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ELISA quantification:
