Cleaved-F10 (I235) Antibody
Description
Overview of Cleaved-F10 (I235) Antibody
The Cleaved-F10 (I235) Antibody is a polyclonal rabbit-derived antibody designed to detect the cleaved form of Factor Xa, a critical enzyme in the coagulation cascade. It specifically targets the heavy chain fragment generated by proteolytic cleavage adjacent to isoleucine 235 (I235) during Factor X activation . This antibody is widely used in research applications, including Western Blot (WB) and Enzyme-Linked Immunosorbent Assay (ELISA), to study coagulation pathways and inflammatory responses .
Mechanism of Action
Factor Xa is a vitamin K-dependent serine protease that converts prothrombin to thrombin in the presence of calcium ions and phospholipids. Its activation involves proteolytic cleavage by Factor VIIa (extrinsic pathway) or Factor IXa (intrinsic pathway), resulting in a 39 kDa heavy chain fragment . The Cleaved-F10 (I235) Antibody recognizes this specific cleavage site, enabling precise detection of activated Factor Xa in experimental models .
Functional Implications
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Coagulation: Central role in thrombin generation and blood clot stabilization .
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Inflammation: Triggers pro-inflammatory signaling via protease-activated receptors (PARs), inducing cytokine release (e.g., IL-6, TNF-α) and adhesion molecule expression .
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Pathophysiology: Linked to cardiovascular diseases, cancer metastasis, and wound healing .
Product Comparisons
The following table highlights key features of commercially available Cleaved-F10 (I235) Antibodies:
Research Applications
This antibody is instrumental in studying:
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Coagulation Pathways: Monitoring Factor X activation in thrombosis models .
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Inflammation: Investigating PAR-mediated signaling in endothelial cells .
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Cancer Research: Analyzing Factor Xa’s role in tumor progression and metastasis .
Example Protocol (WB)
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Sample Preparation: Lyse cells in RIPA buffer with protease inhibitors.
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Electrophoresis: Resolve proteins on 12% SDS-PAGE gels.
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Transfer/Blocking: Transfer to PVDF membranes and block with 5% milk in TBS-T.
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Primary Antibody: Incubate with Cleaved-F10 (I235) Antibody (1:1,000) at 4°C overnight.
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Detection: Use HRP-conjugated secondary antibodies and ECL reagents .
Product Specs
Target Background
- A study showed that an antidote could effectively neutralize the anticoagulant effects of both FXa inhibitors. This suggests that combining drugs and aptamers targeting the same molecule can lead to more specific and potent effects compared to using either agent alone. (PMID: 29863725)
- Research indicates that small vesicles promote the activation of FX by the extrinsic tenase significantly better than large vesicles. (PMID: 28935233)
- miR-24 was found to be overexpressed in major trauma-induced coagulopathy (TIC) patients. The negative correlation between miR-24 and FX suggests that miR-24 may inhibit FX synthesis during TIC. (PMID: 28694557)
- Zymogen-like factor Xa variants exhibit conformational flexibility, and ligands such as its cofactor, factor Va, stabilize the molecule, restoring procoagulant activity. These variants, in the presence of factor Va, serve as effective prohemostatic agents at the site of vascular injury. (PMID: 28692575)
- Data indicates that oxidized lipid vesicles containing phosphatidylserine/polyunsaturated fatty acids promote the inactivation of the ZPI-PZ complex or free ZPI. Binding of PZ-complexed or free ZPI to oxidized vesicles mediates ZPI inactivation (ZPI = protein Z-dependent protease inhibitor; PZ = protein Z; FXa = factor Xa). Blocking the heparin-binding site on ZPI interferes with its binding to lipid or PZ. (PMID: 28717005)
- PTX2 was identified as a novel partner for FX, and both proteins cooperate to prevent their SR-AI-mediated uptake by macrophages. (PMID: 28213380)
- Annexin A2 contributes to lung injury and fibrotic disease by mediating the fibrogenic actions of FXa. (PMID: 28283478)
- A family from Argentina with factor X deficiency displayed a compound heterozygous proband with a combination of a novel mutation and a known one, along with homozygous children. (PMID: 27031279)
- Studies analyze how physiological concentrations of Tissue factor pathway inhibitor inhibit FXa. (PMID: 26607136)
- Compounds 1a, 1g, and 1s exhibited significant FXa inhibitory activity and excellent selectivity over thrombin in in vitro inhibition studies. (PMID: 27089317)
- A study assessed the spectrum of factor X gene mutation in Iranian patients with congenital factor X deficiency (FXD). The majority of mutations were missense mutations, but splice-site mutations were relatively common. (PMID: 26891460)
- The Ala275Val substitution is a pathogenic mutation causing inherited FX deficiency. (PMID: 26708756)
- A homozygous mutation, g.27881G>A(p.Val298Met), in the F10 gene has been identified, likely responsible for low FX concentrations in a specific pedigree. (PMID: 27264807)
- The carboxyl-terminal region of FX downstream of residue K467 is not essential for secretion and contributes modestly to procoagulant properties. (PMID: 26083275)
- In a medical center, rivaroxaban concentrations could be assessed using a rapid chromogenic method. (PMID: 26058941)
- FXa may inhibit lipopolysaccharide-mediated expression of sPLA2-IIA by suppressing cytosolic phospholipase A2 and extracellular signal-regulated kinase 1/2. (PMID: 25399323)
- A family displayed a c.112 G>C mutation in exon 2 of the F10 gene, suggesting that the amino acid substitution may affect the properties of the factor X protein, despite in-silico analysis predicting it as benign. (PMID: 25803519)
- Various acylcarnitines inhibited factor Xa-initiated clotting. (PMID: 26175037)
- Asymmetric processing of mutant factor X Arg386Cys reveals differences between intrinsic and extrinsic pathway activation. (PMID: 26012870)
- The model of human prothrombinase provides a valuable resource for contextualizing previous data and designing future experiments. (PMID: 25153592)
- Factor Xa plasma levels were higher in shift work nurses compared to daytime working nurses. (PMID: 25743687)
- Asp-185 deletion in FX predisposes FX deficient patients to a mild bleeding phenotype. The catalytic activity of the recombinant mutant protease is significantly impaired. (PMID: 25179519)
- Factor Xa plays a role in inhibiting HMGB1-induced septic responses in human umbilical vein endothelial cells and mice. (PMID: 25007770)
- Studies identified procoagulant, tissue factor-bearing microparticles in bronchoalveolar lavage of interstitial lung disease patients. (PMID: 24777000)
- A study demonstrated the clinical utility of monitoring rivaroxaban levels through measurements of anti-Xa activity. (PMID: 25688138)
- The results suggest that the mutation FX-M402T may cause a secretion defect and a molecular abnormality in FX. (PMID: 25064371)
- Prothrombin is proteolytically converted by factor Xa to the active protease thrombin in a reaction accelerated >3,000-fold by cofactor Va. (PMID: 24821807)
- High FXa expression is associated with vascular inflammation in sickle cell disease. (PMID: 24449213)
- Factor Xa induces inflammatory signaling by activating protease-activated receptors in human atrial tissue. (PMID: 24041930)
- Protein Z/protein Z-dependent protease inhibitor and Fxa expression in human gastric cancer cells indicate that these proteins may play a role in anticoagulant events at the tumor tissue. (PMID: 24158387)
- The structure of factor Xa is regulated by factor Va and phosphatidylserine. (PMID: 24467409)
- Factor X deficiency is associated with bleeding due to poor recognition of the mutant substrate by Factor IXa. (PMID: 23677006)
- In carotid artery plaque, expression of SPHK1 was observed at smooth muscle cell-rich sites and was co-localized with intraplaque FX/FXa content. (PMID: 23658376)
- Seven missense mutations were identified in the F10 of four probands with FX deficiency, six of which (Ser425Pro, Ala-29Pro, Phe324Leu, Ala235Thr, Cys111Arg, and Met362Thr) were novel and associated with type I FX deficiency. (PMID: 23664564)
- Anti-FXa antithrombin assay is recommended as a first-line test to detect type II heparin-binding site antithrombin deficiency. (PMID: 24124146)
- A novel function for AT was discovered, which accelerates the modulation of FXa into the fibrinolytic form. (PMID: 23416531)
- Despite a delay in reaching therapeutic anti-FXa levels on unfractionated heparin treatment, infants monitored with the adult-based anti-FXa range showed a high thrombus resolution rate, no thrombus progression, but a relatively high bleeding rate. (PMID: 22244010)
- Two novel causative mutations of the Factor 10 gene were identified in a Chinese proband with severe Factor X deficiency and mild clinical symptoms. (PMID: 22931370)
- The Kunitz 1 and Kunitz 3 domains of tissue factor pathway inhibitor are essential for efficient inhibition of factor Xa. (PMID: 22627666)
- Research suggests that FX binds to the surface of human species C adenovirus and becomes a pathogen-associated molecular pattern, triggering activation of innate immunity upon viral entry into the cell. (PMID: 23019612)
- Three unrelated Palestinian patients were found to be homozygous for c302delG, a novel frameshift mutation in the F10 gene causing a stop codon at amino acid 73. (PMID: 22008904)
- srxA and prxA (2-Cys peroxiredoxin) genes are induced in response to oxidative stress. (PMID: 21651559)
- Patients with hypomethylated F10 promoter in tumors had shorter median overall survival. (PMID: 22160665)
- RXA plasma levels can be quantified accurately and precisely by a chromogenic anti-FXa assay on different coagulometers in various laboratories. (PMID: 21840043)
- Localization of PZ/ZPI and FX in colon cancer cells indicates that PZ/ZPI may contribute to anticoagulant events at the tumor site. (PMID: 22424030)
- Alboserpin is an atypical serpin that targets FXa and displays unique phospholipid specificity. (PMID: 21673107)
- The regulatory action of FXa on PAR-2 was concentration-dependent and mimicked by a PAR-2-selective activating peptide. (PMID: 21871560)
- Murine and human factor X exhibit differential effects on adenovirus transduction via cell-surface heparan sulfate. (PMID: 21596747)
- Rivaroxaban can be determined by different factor Xa-specific chromogenic substrate assays, leading to reduced interassay variability. (PMID: 21811937)
- Six FXIa catalytic domain residues (Glu(98), Tyr(143), Ile(151), Arg(3704), Lys(192), and Tyr(5901)) were subjected to mutational analysis to investigate interactions between FXIa and a synthetic substrate, the substrate factor IX, and inhibitor PN2KPI. (PMID: 21778227)
Q&A
What is the Cleaved-F10 (I235) Antibody and what epitope does it specifically recognize?
The Cleaved-F10 (I235) Antibody is a rabbit polyclonal antibody that specifically detects endogenous levels of activated Factor Xa heavy chain protein fragments resulting from cleavage adjacent to isoleucine-235 . This antibody was generated using a synthesized peptide derived from human Factor X (FA10) corresponding to amino acids 216-265 . It provides a valuable tool for researchers specifically studying the activated form of coagulation factor X, which plays a critical role in the blood coagulation cascade by converting prothrombin to thrombin .
What experimental applications has this antibody been validated for?
The Cleaved-F10 (I235) Antibody has been validated primarily for Western Blot (WB) and ELISA applications . For Western Blot analysis, the recommended dilution range is 1:500-1:2000, while ELISA applications typically use a more dilute concentration of approximately 1:20000 . The antibody has demonstrated consistent results across these applications when used with appropriate controls and optimization protocols. Researchers should validate the antibody in their specific experimental systems as performance can vary depending on sample preparation methods and experimental conditions.
What species reactivity has been confirmed for this antibody?
This antibody has been experimentally validated to react with human, mouse, and rat samples . This cross-species reactivity makes the antibody particularly valuable for comparative studies and translational research between animal models and human samples. The conservation of the target epitope across these species suggests the functional importance of this region in Factor X biology.
What are the optimal sample preparation methods for detecting cleaved Factor X?
For optimal detection of cleaved Factor X using this antibody, researchers should implement the following methodological approaches:
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Tissue/Cell Lysates: Use lysis buffers containing protease inhibitor cocktails to prevent artificial proteolytic processing during sample preparation. Cold extraction conditions (4°C) should be maintained throughout the procedure.
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Blood/Plasma Samples: Process samples immediately after collection with appropriate anticoagulants (sodium citrate preferred for coagulation studies). Centrifuge at 2000-3000 × g for 15 minutes at 4°C to separate plasma.
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Storage Considerations: Aliquot samples to avoid freeze-thaw cycles and store at -80°C for long-term preservation of protein integrity .
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Denaturation Conditions: For Western Blot applications, mild denaturation conditions are recommended to preserve the epitope structure recognized by the antibody.
How should Western Blot protocols be optimized for this antibody?
To achieve optimal results with the Cleaved-F10 (I235) Antibody in Western Blot applications, researchers should consider the following methodological recommendations:
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Sample Loading: Load 20-50 μg of total protein per lane, with appropriate positive and negative controls.
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Transfer Conditions: Use PVDF membranes (0.45 μm pore size) with wet transfer systems for optimal protein transfer.
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Blocking: Block membranes with 5% non-fat dry milk or BSA in TBST for 1-2 hours at room temperature.
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Primary Antibody Incubation: Dilute antibody 1:500-1:2000 in blocking buffer and incubate overnight at 4°C with gentle agitation .
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Secondary Antibody: Use HRP-conjugated anti-rabbit IgG secondary antibody at 1:5000-1:10000 dilution.
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Detection System: ECL-based detection systems provide sufficient sensitivity for most applications.
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Expected Band Size: The cleaved Factor Xa heavy chain appears at approximately 39 kDa .
What controls should be included when using this antibody in experimental protocols?
A robust experimental design using the Cleaved-F10 (I235) Antibody should include the following controls:
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Positive Controls: Include samples known to contain activated Factor X, such as:
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Negative Controls:
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Samples from Factor X-deficient models
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Antibody preincubation with the immunizing peptide (216-265 aa region)
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Primary antibody omission control
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Processing Controls:
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Paired samples of activated versus non-activated coagulation cascades
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Time-course activation samples to demonstrate progressive cleavage
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These controls help validate antibody specificity and provide benchmarks for interpreting experimental results.
How can researchers distinguish between cleaved and uncleaved Factor X in experimental samples?
Distinguishing between cleaved and uncleaved Factor X requires careful analytical approaches:
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Molecular Weight Analysis: Uncleaved Factor X appears at approximately 54.7 kDa, while the cleaved activated heavy chain migrates at approximately 39 kDa in SDS-PAGE gels .
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Parallel Analysis: Run the same samples with both cleaved-specific (I235) antibody and antibodies recognizing total Factor X to determine the proportion of activated versus zymogen forms.
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Biochemical Validation: Correlate immunological detection with functional assays measuring Factor Xa enzymatic activity.
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Densitometric Analysis: Calculate the ratio of cleaved to uncleaved forms across experimental conditions to quantify activation status.
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Non-reducing vs. Reducing Conditions: Compare electrophoretic mobility under both conditions to distinguish disulfide-linked fragments.
What methodological considerations are important when analyzing Factor X activation in disease models?
When investigating Factor X activation in disease models using this antibody, researchers should consider:
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Temporal Dynamics: Coagulation cascades have rapid kinetics; time-course experiments are essential for capturing relevant activation events.
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Spatial Distribution: In tissue sections, the localization of cleaved Factor X may provide insights into microenvironmental activation, particularly at invasive fronts of tumors .
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Contextual Analysis: Factor X activation should be interpreted in the context of other coagulation factors and inhibitors present in the sample.
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Quantitative Assessment: Develop standardized quantification methods (such as densitometry for Western Blots or standardized ELISA procedures) to enable cross-experiment comparisons.
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Correlation with Clinical Parameters: In translational research, correlate cleaved Factor X levels with disease severity, progression, or treatment response.
How can the Cleaved-F10 (I235) Antibody be used to investigate non-hemostatic functions of Factor Xa?
Beyond its canonical role in coagulation, Factor Xa participates in cellular signaling and inflammation. Advanced research applications include:
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Receptor-Mediated Signaling: Use the antibody to investigate Factor Xa interactions with protease-activated receptors (PARs) through co-immunoprecipitation or proximity ligation assays.
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Pro-inflammatory Pathway Analysis: Combine with phosphorylation-specific antibodies to map signaling cascades activated by cleaved Factor X in target cells .
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Cell-Specific Activation: Employ flow cytometry with permeabilization protocols to quantify intracellular cleaved Factor X in specific cell populations.
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Subcellular Localization: Utilize immunofluorescence confocal microscopy with the Cleaved-F10 (I235) Antibody to identify non-canonical subcellular localization patterns.
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Protein-Protein Interaction Networks: Apply mass spectrometry following immunoprecipitation to identify novel interaction partners of activated Factor X.
What approaches can be used to study Factor X in tumor microenvironments?
Investigating Factor X in tumor biology presents unique research opportunities:
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Tumor Invasion Analysis: Study the co-localization of cleaved Factor X with markers of tumor invasion at the tumor-stromal interface .
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Protease Network Mapping: Investigate proteolytic networks by combining detection of cleaved Factor X with other proteases present in tumor microenvironments.
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Engineered Antibody Studies: Compare cleavage patterns of therapeutic antibodies in the presence of activated Factor X to develop protease-resistant antibody variants .
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Ex Vivo Tissue Analysis: Apply the antibody in fresh tumor explant cultures to monitor real-time Factor X activation in response to experimental interventions.
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Microenvironmental Correlation: Correlate Factor X activation with hypoxia, acidosis, or inflammatory markers to understand activation triggers.
How can researchers investigate the relationship between vitamin K-dependent modifications and Factor X cleavage?
The vitamin K-dependent nature of Factor X presents interesting research questions that can be addressed using this antibody:
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Comparative Analysis: Compare detection of cleaved Factor X between normal and vitamin K-deficient samples to assess the impact on proteolytic processing.
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Post-translational Modification Studies: Combine the Cleaved-F10 (I235) Antibody with antibodies targeting γ-carboxyglutamic acid residues to investigate the relationship between carboxylation and cleavage efficiency .
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Warfarin Effect Studies: Examine samples from warfarin-treated subjects to determine how vitamin K antagonism affects the generation of the I235 cleaved fragment.
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Calcium-Dependent Processing: Analyze Factor X cleavage in the presence of varying calcium concentrations, as calcium binding is facilitated by vitamin K-dependent carboxylation .
What are common technical challenges when using the Cleaved-F10 (I235) Antibody and how can they be addressed?
| Challenge | Possible Causes | Methodological Solutions |
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| High background | Non-specific binding; Insufficient blocking | Increase blocking time to 2-3 hours; Use 5% BSA instead of milk; Increase washing duration and volume |
| Weak signal | Low target abundance; Inefficient transfer | Increase antibody concentration (1:500); Extend primary antibody incubation to 24-48 hours at 4°C; Use enhanced chemiluminescence substrate |
| Multiple bands | Sample degradation; Cross-reactivity | Use fresh samples with complete protease inhibitor cocktails; Validate with blocking peptide competition |
| Inconsistent results | Variable Factor X activation | Standardize activation conditions; Include time-course controls |
| No signal | Epitope masking; Sample preparation issues | Try different epitope retrieval methods; Verify protein extraction with total Factor X antibodies |
What storage conditions ensure optimal stability and performance of this antibody?
To maintain optimal activity of the Cleaved-F10 (I235) Antibody, researchers should follow these evidence-based storage recommendations:
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Long-term Storage: Store at -20°C for up to one year from receipt date .
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Working Stock: For frequent use, a small aliquot can be stored at 4°C for up to one month.
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Formulation: The antibody is typically supplied in PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide, providing stability during freeze-thaw cycles .
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Aliquoting: Upon receipt, prepare small single-use aliquots to avoid repeated freeze-thaw cycles.
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Handling: Briefly centrifuge vials before opening to collect liquid at the bottom of the tube.
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Working Dilutions: Freshly prepared antibody dilutions yield optimal results; avoid storing diluted antibody for extended periods.
