Cleaved-F13A1 (G39) Antibody

What is Cleaved-F13A1 (G39) Antibody and what does it detect?

Cleaved-F13A1 (G39) Antibody is a polyclonal antibody specifically designed to detect endogenous levels of activated Factor XIIIa protein fragments resulting from cleavage adjacent to the G39 position. This antibody recognizes the N-terminal region of Factor XIIIa that becomes exposed following proteolytic activation . Factor XIIIa is the activated form of coagulation factor XIII, which functions at the final stage of the coagulation cascade to stabilize fibrin clots .

The antibody binds specifically to the human F13A1 protein (also known as coagulation factor XIII A chain), which is encoded by the F13A1 gene. This protein serves as a transglutaminase that catalyzes the formation of gamma-glutamyl-epsilon-lysine crosslinks between fibrin molecules, thus strengthening blood clots and preventing further bleeding .

What applications are Cleaved-F13A1 (G39) Antibody validated for?

The Cleaved-F13A1 (G39) Antibody has been validated for several research applications:

For Western Blot applications, the antibody effectively detects the cleaved form of Factor XIIIa at the expected molecular weight range . When using this antibody for new applications or experimental systems, preliminary validation is strongly recommended to ensure optimal results.

How is the Cleaved-F13A1 (G39) Antibody produced and purified?

The Cleaved-F13A1 (G39) Antibody is produced through immunization of rabbits with a synthesized peptide derived from the N-terminal region of human Factor XIIIa . The immunogen specifically targets the amino acid range 20-69 of the human F13A1 protein .

The purification process involves:

• Collection of rabbit antiserum containing polyclonal antibodies

• Affinity chromatography using the epitope-specific immunogen

• Quality control testing for specificity and sensitivity

• Formulation in PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide

This rigorous production and purification methodology ensures high specificity for the cleaved form of Factor XIIIa while minimizing cross-reactivity with other proteins.

What controls should be used when working with Cleaved-F13A1 (G39) Antibody?

When designing experiments with Cleaved-F13A1 (G39) Antibody, appropriate controls are essential for result validation:

• Positive Control: Human tissue or cell lysates known to express Factor XIIIa, such as platelets, macrophages, or liver tissue samples

• Negative Control: Samples from F13A1 knockout models or cell lines with confirmed absence of F13A1 expression

• Peptide Competition Assay: Pre-incubation of the antibody with the immunizing peptide to confirm specificity

• Loading Control: Use of housekeeping proteins (β-actin, GAPDH) for Western Blot normalization

• Secondary Antibody Control: Omitting primary antibody to assess non-specific binding

Including these controls helps ensure experimental validity and supports accurate interpretation of results when investigating Factor XIIIa activation in various physiological and pathological contexts.

How can I validate the specificity of Cleaved-F13A1 (G39) Antibody in my experimental system?

Validating antibody specificity is critical for obtaining reliable research data. For Cleaved-F13A1 (G39) Antibody, consider implementing these advanced validation strategies:

• Genetic Validation: Apply CRISPR-Cas9 gene editing to knockout F13A1 in your cell line of interest. Compare antibody reactivity between wild-type and knockout cells via Western blot or immunostaining .

• Independent Antibody Approach: Use two antibodies that recognize different epitopes of F13A1. The staining or detection pattern should be identical if both antibodies are specific . For example:

  • Cleaved-F13A1 (G39) polyclonal antibody

  • An alternative monoclonal antibody like F13A1 (OTI9E2)

• Recombinant Expression System: Express tagged recombinant F13A1 protein in a cell line with low endogenous expression. Confirm co-localization of the antibody signal with the tag detection .

• Application-Specific Validation:

  • For Western blot: Verify the molecular weight (~83 kDa for full-length F13A1, with cleaved fragments at expected sizes)

  • For immunoprecipitation: Perform mass spectrometry on immunoprecipitated samples to confirm identity

  • For IHC: Compare staining patterns with published literature on F13A1 expression in specific tissues

Remember that validation must be performed for each specific application and sample type, as antibody performance can vary across different experimental conditions .

What methodological considerations are important when studying Factor XIIIa activation using Cleaved-F13A1 (G39) Antibody?

Studying Factor XIIIa activation requires careful experimental design:

• Sample Preparation Considerations:

  • Blood collection: Use citrate anticoagulant rather than EDTA or heparin to preserve Factor XIII integrity

  • Timing is critical: Rapid processing prevents artificial activation

  • Protease inhibitors: Include specific inhibitors to prevent ex vivo proteolysis

  • Activation conditions: Factor XIII requires thrombin cleavage and calcium for activation

• Distinguishing Native vs. Activated Forms:

  Form	Molecular Configuration	Detection Approach
  Zymogen Factor XIII	Heterotetramer (A₂B₂)	Use antibodies against intact A subunit
  Activated Factor XIIIa	Free catalytic A subunits	Use Cleaved-F13A1 (G39) Antibody
  Platelet Factor XIII	Homodimer (A₂)	Different extraction protocol required

• Activation Protocol Optimization:

  • Thrombin concentration: Titrate to achieve reproducible activation

  • Calcium concentration: Physiological levels (2-2.5 mM) are optimal

  • Time course analysis: Establish activation kinetics in your specific model

  • Temperature: Perform reactions at 37°C to mimic physiological conditions

• Confirmation of Activation:

  • Activity assays: Measure transglutaminase activity using specific substrates

  • Correlate activity measurements with antibody detection of cleaved forms

These methodological considerations enhance the reliability of Factor XIIIa activation studies and ensure accurate interpretation of results obtained with the Cleaved-F13A1 (G39) Antibody.

How can Cleaved-F13A1 (G39) Antibody be used to investigate Factor XIII polymorphisms and their clinical implications?

The F13A1 gene contains several important polymorphisms with potential clinical relevance. The Cleaved-F13A1 (G39) Antibody can be utilized to investigate these variants through carefully designed experiments:

• Val34Leu (V34L) Polymorphism Studies:
  The common Val34Leu polymorphism affects the rate of Factor XIII activation by accelerating the process . Research protocols should include:

  • Genotyping subjects for Val34Leu polymorphism

  • Comparing Factor XIIIa activation kinetics using Cleaved-F13A1 (G39) Antibody in Western blots at multiple time points

  • Quantitative analysis of activated Factor XIIIa levels in different genotype groups

  • Correlating activation patterns with clinical parameters (clot structure, bleeding risk, cardiovascular outcomes)

• Experimental Design for Polymorphism Research:

  Parameter	Val/Val (Wild-type)	Val/Leu (Heterozygous)	Leu/Leu (Homozygous)
  Activation Rate	Baseline	Intermediate	Accelerated
  Sample Time Points	0, 5, 10, 15, 30 min	0, 2, 5, 10, 15 min	0, 1, 2, 5, 10 min
  Thrombin Concentration	Standard (1 U/ml)	May require adjustment	May require adjustment
  Expected Findings	Reference pattern	Earlier detection of cleaved form	Significantly earlier detection

• Methodological Approaches:

  • Develop a multiplexed assay combining Cleaved-F13A1 (G39) Antibody with other markers of coagulation

  • Use patient-derived samples with known genotypes to establish activation profiles

  • Implement densitometric analysis of Western blots to quantify activation rates

  • Consider developing a modified ELISA protocol using Cleaved-F13A1 (G39) Antibody to quantify activation products

• Interpreting Complex Results:

  • Consider the context of other coagulation factors when analyzing data

  • Account for environmental factors that may influence Factor XIII activation

  • Establish reference ranges for different genotype groups in your experimental system

This research approach enables the investigation of how genetic variations in F13A1 affect protein function, potentially contributing to our understanding of thrombotic and hemorrhagic disorders .

What are effective troubleshooting strategies for optimizing Cleaved-F13A1 (G39) Antibody performance in Western blot applications?

When optimizing Western blot protocols with Cleaved-F13A1 (G39) Antibody, researchers may encounter several challenges. Below are evidence-based troubleshooting strategies:

• Weak or No Signal Issues:

  • Verify protein loading: 30-50 μg total protein is typically sufficient

  • Optimize antibody concentration: Test a dilution series from 1:500 to 1:2000

  • Extend incubation time: Try overnight incubation at 4°C

  • Enhance detection: Consider using high-sensitivity ECL substrates

  • Reduce washing stringency: Decrease salt concentration or detergent in TBST

• Multiple or Non-specific Bands:

  • Increase blocking duration: Try 1-2 hours with 5% non-fat milk or BSA

  • Adjust antibody dilution: Use more diluted antibody (1:1000-1:2000)

  • Add detergent: Include 0.1-0.2% Tween-20 in antibody dilution buffer

  • Modified blocking agent: Try different blockers (casein, commercial blockers)

  • Protein extraction optimization: Ensure complete denaturation with appropriate buffer

• Specific Protocol Modifications for Factor XIIIa Detection:

  Issue	Potential Solution	Scientific Rationale
  Detecting both zymogen and activated forms	Use gradient gels (4-15%)	Better separation of closely sized proteins
  Inconsistent activation state	Include thrombin/CaCl₂ treatment controls	Standardizes activation state
  Cross-reactivity with Factor XIII B	Use reducing conditions	Disrupts A₂B₂ tetramer structure
  Degradation during preparation	Add specific protease inhibitors	Prevents artificial proteolysis
  Background in plasma samples	Pre-clear with Protein A/G beads	Removes potentially cross-reactive proteins

• Advanced Optimization Techniques:

  • Consider native vs. reducing conditions based on your research question

  • For cleaved form detection, optimize sample preparation to preserve fragmentation pattern

  • Implement quantitative Western blot techniques with appropriate loading controls

  • Consider pre-incubation of antibody with non-specific proteins (e.g., cell lysate from non-expressing cells)

Implementing these strategies systematically can significantly improve detection specificity and sensitivity when using Cleaved-F13A1 (G39) Antibody in Western blot applications.

How can Cleaved-F13A1 (G39) Antibody be integrated into multiparameter analysis of coagulation disorders?

Incorporating Cleaved-F13A1 (G39) Antibody into comprehensive coagulation studies requires strategic experimental design. Here's a methodological framework for multiparameter analyses:

• Integrated Coagulation Cascade Assessment:
  Create a panel of key coagulation markers that includes:

  Coagulation Phase	Key Markers	Analysis Method	Role of Cleaved-F13A1 (G39) Antibody
  Initiation	Tissue Factor, Factor VII	Flow cytometry, ELISA	Baseline comparison
  Amplification	Thrombin, Factor X	Chromogenic assays	Activation trigger monitoring
  Propagation	Fibrinogen, Factor V	Functional assays	Context for XIII activation
  Stabilization	Factor XIIIa, PAI-1	Western blot, ELISA	Direct measurement of cleaved XIIIa
  Fibrinolysis	D-dimer, Plasmin	Immunoassays	Correlation with clot stability

• Sequential Sampling Methodology:

  • Establish standardized time points for sample collection

  • Include parallel assessment of Factor XIIIa activity and cleaved form detection

  • Correlate antibody-detected cleaved forms with functional transglutaminase activity

  • Implement paired analysis of pre- and post-activation samples

• Bioimaging Applications:

  • Develop dual-labeling protocols combining Cleaved-F13A1 (G39) Antibody with other coagulation markers

  • Optimize immunofluorescence protocols for tissue sections and thrombi

  • Establish co-localization analysis parameters for Factor XIIIa with fibrin and platelets

  • Consider super-resolution microscopy for detailed structural analysis of cross-linked fibrin networks

• Clinical Sample Analysis Considerations:

  • Standardize pre-analytical variables (collection, processing, storage)

  • Establish reference ranges for different patient populations

  • Implement batch controls to minimize inter-assay variability

  • Develop analysis algorithms that integrate Factor XIIIa data with other coagulation parameters

This integrated approach provides a more comprehensive understanding of coagulation disorders by positioning Factor XIIIa activation within the broader context of the coagulation cascade, offering insights into both basic mechanisms and potential therapeutic interventions.

What emerging research areas can benefit from Cleaved-F13A1 (G39) Antibody applications?

The Cleaved-F13A1 (G39) Antibody has potential applications in several cutting-edge research areas beyond traditional coagulation studies:

• Cardiovascular Disease Mechanisms: Investigating the role of Factor XIIIa in atherosclerotic plaque stability, post-myocardial infarction remodeling, and thrombosis risk stratification .

• Wound Healing and Tissue Regeneration: Examining Factor XIIIa's function in extracellular matrix organization, cell migration, and angiogenesis during tissue repair processes .

• Cancer Biology: Exploring the involvement of Factor XIIIa in tumor microenvironment organization, metastatic potential, and interaction with the extracellular matrix.

• Neurodegenerative Disorders: Studying potential roles of Factor XIIIa in blood-brain barrier integrity, microglial function, and neuroinflammatory processes.

• Biomarker Development: Utilizing cleaved Factor XIIIa detection in developing predictive biomarkers for thrombotic events or monitoring anticoagulant therapy efficacy.

Each of these research areas presents unique opportunities for applying Cleaved-F13A1 (G39) Antibody in novel experimental paradigms, potentially leading to important discoveries in disease mechanisms and therapeutic approaches.

How should researchers evaluate and report Cleaved-F13A1 (G39) Antibody data in publications?

To enhance reproducibility and scientific rigor when publishing research using Cleaved-F13A1 (G39) Antibody, consider these best practices:

• Comprehensive Antibody Documentation:

  • Report complete antibody information: catalog number, manufacturer, lot number, species reactivity

  • Describe validation methods used to confirm specificity in your experimental system

  • Document dilutions, incubation conditions, and detection methods

• Experimental Controls Documentation:

  • Detail all positive and negative controls included

  • Explain knockdown/knockout validation if performed

  • Describe peptide competition assays or other specificity tests

• Quantification and Statistical Analysis:

  • Clearly explain normalization methods used for quantitative analyses

  • Include appropriate statistical tests with justification

  • Present raw data alongside normalized results when possible

  • Consider including supplementary materials showing full blots or staining patterns

• Methodology Limitations Acknowledgment:

  • Discuss potential cross-reactivity considerations

  • Address limitations in detecting specific activation states

  • Acknowledge any inconsistencies observed during experiments