HGFAC Antibody

What is HGFAC and what is its primary biological function?

HGFAC (Hepatocyte Growth Factor Activator) is a trypsin-like S1 family serine endopeptidase that plays a critical role in tissue regeneration by converting single-chain inactive proHGF to mature two-chain active HGF. Phylogenetically, HGFAC evolved alongside the coagulation and fibrinolysis systems, with evidence indicating that the gene for factor XII evolved from duplication of the HGFAC gene . This evolutionary relationship suggests HGFAC's primary biological function is responding to tissue injuries. In its active form, HGFAC initiates a signaling cascade through HGF-MET pathways that promotes tissue repair and regeneration, with particularly important roles in liver, kidney, and intestinal injury models .

What are the technical specifications of typical HGFAC antibodies used in research?

Research-grade HGFAC antibodies typically present the following specifications:

When selecting an HGFAC antibody, researchers should confirm the peptide region used as immunogen, as this affects which domains or conformational states of the protein will be recognized .

How should HGFAC antibody specificity be validated prior to experimental use?

Proper validation of HGFAC antibody specificity requires a multi-step approach:

• Western blot analysis: Confirm single band detection at the expected molecular weight (approximately 70 kDa) using positive control samples such as K562 or Jurkat cell extracts .

• Negative controls: Include tissues or cell lines known to lack HGFAC expression to rule out non-specific binding.

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide to confirm signal elimination in positive samples.

• Cross-reactivity assessment: If planning cross-species studies, validate each intended species separately as sequence homology doesn't guarantee equivalent binding efficiency.

• Knockout/knockdown validation: When available, tissue or cell samples with genetic deletion or suppression of HGFAC provide the gold standard for specificity confirmation.

For optimal western blot results, initial dilution ranges of 1:500-1:2000 are recommended for most HGFAC antibodies, with optimization required for each specific experimental system .

What tissue samples and experimental conditions are optimal for HGFAC antibody applications?

For optimal HGFAC antibody applications, researchers should consider:

Tissue samples: Liver tissue represents the primary site of HGFAC production and shows high expression levels, making it an ideal positive control. Additionally, serum samples can be used as HGFAC is secreted into circulation . When working with liver serum samples, appropriate sample preparation techniques are essential to minimize interference from abundant serum proteins.

Experimental models: Several validated injury models demonstrate robust HGFAC activation:

• CCl₄-induced liver injury

• HgCl₂-induced renal injury

• Acetic acid-induced colitis

• Dextran sodium sulfate-induced colitis

These models show significant upregulation of HGFAC activity specifically at injury sites, making them valuable for studying physiological HGFAC functions.

Activation conditions: When studying HGFAC activation mechanisms, include thrombin in experimental setups, particularly in the presence of glycosaminoglycans such as dextran sulfate, chondroitin sulfate, or heparin, which significantly enhance activation efficiency .

For sample preparation, minimize freeze-thaw cycles as repeated cycles can reduce antibody reactivity and compromise experimental outcomes .

How can researchers distinguish between inactive proHGFAC and active HGFAC in experimental samples?

Distinguishing between inactive proHGFAC and active HGFAC requires specialized methodological approaches:

• Molecular weight analysis: ProHGFAC and active HGFAC have distinct molecular weights that can be differentiated by high-resolution western blotting. ProHGFAC appears as a single-chain molecule, while active HGFAC presents as a two-chain structure linked by disulfide bonds .

• Activity-based assays: Utilize synthetic peptide substrates containing HGFAC cleavage sites coupled with fluorogenic or chromogenic reporters to measure enzymatic activity directly.

• Conformation-specific antibodies: Some antibodies specifically recognize the active conformation of HGFAC, which undergoes significant structural changes upon activation.

• Thrombin-activation experiments: Compare samples before and after treatment with thrombin (a physiological activator of proHGFAC) to confirm the identity of the inactive zymogen versus active protease .

• Inhibitor complex formation: Active HGFAC forms complexes with endogenous inhibitors like HAI-1/SPINT1, which can be detected using co-immunoprecipitation or western blotting under non-reducing conditions .

For optimal detection sensitivity, researchers should consider using antibodies targeting different domains of HGFAC to distinguish between its various processed forms and activation states.

What are the recommended protocols for applying HGFAC antibodies in tissue regeneration models?

When implementing HGFAC antibodies in tissue regeneration models, researchers should follow these methodological approaches:

• Injury model selection: Use established models that demonstrate robust HGFAC activation, such as CCl₄-induced liver injury or dextran sodium sulfate-induced colitis . These models provide reproducible injury patterns for studying HGFAC's regenerative functions.

• Timing considerations: Design tissue collection time points that capture both immediate post-injury activation (6-24 hours) and subsequent regenerative processes (3-14 days), as HGFAC activation patterns differ temporally across these phases .

• Western blot protocol optimization:

  • Sample preparation: Use RIPA buffer supplemented with protease inhibitors

  • Loading: 20-50μg total protein per lane

  • Dilution: Start with 1:500-1:2000 antibody dilution and optimize

  • Blocking: 5% non-fat milk or BSA in TBST (1-2 hours)

  • Incubation: Overnight at 4°C for primary antibody

  • Detection: HRP-conjugated secondary antibody with enhanced chemiluminescence

• Comparative analysis: Include both injured and non-injured tissues from the same animal to establish baseline expression and activation levels.

• Inhibition studies: Include experimental groups receiving neutralizing anti-HGFAC antibodies to establish the specific contribution of HGFAC to the observed regenerative effects .

• Cross-validation: Complement protein detection with mRNA analysis (RT-qPCR) to assess both transcriptional and post-translational regulation of HGFAC during regeneration.

This systematic approach ensures comprehensive characterization of HGFAC's role in tissue regeneration mechanisms.

How does HGFAC activation influence stem cell behavior in regenerative processes?

HGFAC activation represents a critical signaling mechanism that influences stem cell behavior during tissue regeneration through multiple pathways:

HGFAC functions as an alarmin upon tissue injury, transmitting systemic signals that prime stem cells for enhanced regenerative capacity. Recent research by Rodgers et al. demonstrated that activated circulating HGFAC in plasma following experimental tissue injury in mice mediates a crucial signaling cascade to distant, non-injured tissues . This signaling induces quiescent muscle stem cells and fibro-adipogenic progenitors to transition from the G₀ state to a primed GAlert state through HGF-MET-mTORC1 signaling pathways .

The molecular mechanism involves:

• Tissue injury triggering HGFAC release into circulation

• Systemic distribution of activated HGFAC

• Activation of pericellular proHGF in stem cell niches

• HGF binding to MET receptors on quiescent stem cells

• Activation of mTORC1 signaling

• Transition from G₀ to GAlert state

This priming effect significantly enhances the regenerative capacity of stem cells, preparing them for rapid response to subsequent injury signals. Evidence suggests this mechanism extends beyond muscle stem cells to other MET-expressing stem cell populations, including epidermal, mesenchymal, hepatic, and hematopoietic stem cells .

Furthermore, HGFAC activity may complement other alarmin pathways, such as the HMGB1-CXCL12-CXCR4 pathway, as inhibition of MET activity reduces surface CXCR4 expression in muscle stem cells . This suggests interconnected signaling networks that collectively orchestrate enhanced tissue regeneration capacity.

What role do endogenous inhibitors play in regulating HGFAC function during tissue repair?

Endogenous inhibitors play critical regulatory roles in modulating HGFAC activity during tissue repair processes through complex mechanisms:

Two primary endogenous inhibitors regulate HGFAC activity in physiological contexts:

• Protein C Inhibitor (PCI/SERPINA5):

  • Circulating serpin-type inhibitor produced primarily by the liver

  • Also expressed locally in reproductive organs

  • Forms irreversible complexes with HGFAC

  • Functions as systemic regulator of HGFAC activity

• HGFAC Inhibitor Type 1 (HAI-1/SPINT1):

  • Kunitz-type transmembrane inhibitor expressed by epithelial cells

  • Contains two extracellular Kunitz domains (KD1 and KD2)

  • KD1 mediates reversible complex formation with HGFAC

  • Regulates HGFAC in the pericellular microenvironment

The balance between HGFAC and its inhibitors represents a critical regulatory mechanism that ensures appropriate spatial and temporal activation of HGF-MET signaling during tissue repair processes, preventing excessive or inappropriate pathway activation that could lead to fibrosis or neoplastic transformation.

How can researchers effectively use HGFAC antibodies to study inflammatory mechanisms in tissue injury?

Researchers can implement several sophisticated approaches using HGFAC antibodies to investigate inflammatory mechanisms in tissue injury:

• Temporal profiling of HGFAC activation:

  • Collect tissue samples at sequential time points post-injury (6h, 24h, 72h, 7d)

  • Perform western blot analysis with HGFAC antibodies to track conversion from proHGFAC to active HGFAC

  • Correlate HGFAC activation patterns with inflammatory cytokine profiles (IL-1, TNF-α)

• Spatial analysis using immunohistochemistry:

  • Co-stain tissue sections with HGFAC antibodies and inflammatory cell markers

  • Analyze proximity of activated HGFAC to inflammatory infiltrates

  • Quantify HGFAC activation in relation to distance from injury epicenter

• Cytokine co-stimulation experiments:

  • Treat cell cultures with inflammatory cytokines (IL-1, TNF-α, etc.)

  • Measure HGFAC activation and HGF mRNA expression changes

  • Use HGFAC antibodies to neutralize activity and assess downstream effects

• Comparative injury models:

  • Implement both inflammatory (CCl₄-induced liver injury) and non-inflammatory (partial hepatectomy) models

  • Compare HGFAC activation patterns between models

  • Research has shown that CCl₄ treatment induces significant proHGF activation in liver tissue, while partial hepatectomy does not induce robust proHGF activation

• Inhibition studies in inflammatory environments:

  • Use neutralizing HGFAC antibodies in injury models

  • Analyze effects on inflammatory cell recruitment and cytokine production

  • Assess impact on tissue repair timeline and outcome quality

The integration of these approaches allows researchers to delineate the complex interplay between inflammation and HGFAC-mediated tissue repair mechanisms, revealing potential intervention targets for enhancing regeneration while minimizing inflammatory damage.

How can HGFAC antibodies be used to study age-related muscle atrophy mechanisms?

Recent research has opened promising avenues for using HGFAC antibodies to investigate and potentially counteract age-related muscle atrophy mechanisms:

Researchers have recently developed specialized antibodies that target specific nitration sites on hepatocyte growth factor (HGF), which is activated by HGFAC. This approach addresses a fundamental mechanism in age-related muscle deterioration: the dysfunction of HGF due to nitration of specific tyrosine residues (the 198th and 250th tyrosine amino acids) .

The methodology involves:

• Identification of nitration-sensitive domains: Using structural analysis to identify critical tyrosine residues (198th and 250th) on HGF that undergo nitration with aging .

• Development of site-specific antibodies: Creating and screening antibodies that specifically bind to these regions to prevent nitration while maintaining normal HGF function .

• Functional validation: Testing candidate antibodies (such as 1H41C10 and 1H42F4N) for their ability to block tyrosine nitration without disrupting HGF's capacity to activate satellite cells .

• Application in age-related muscle models: Using these antibodies to restore HGF function in aged muscle tissue and measure effects on satellite cell activation and muscle regeneration.

Researchers investigating age-related muscle weakness can use these specialized antibodies alongside standard HGFAC antibodies to comprehensively characterize the HGFAC-HGF axis in aging muscle tissue and develop targeted interventions for muscle atrophy.

What methodological approaches are recommended for studying HGFAC's alarmin function?

To effectively investigate HGFAC's emerging role as an alarmin in systemic regenerative responses, researchers should implement the following methodological approaches:

• Remote tissue analysis protocol:

  • Create localized tissue injury in a primary site (muscle, liver, etc.)

  • Collect tissue samples from distant, uninjured sites (contralateral muscle, etc.)

  • Analyze stem cell activation state using cell cycle markers (Ki67, MCM2)

  • Assess mTORC1 pathway activation (phospho-S6, phospho-4E-BP1)

  • Correlate with circulating HGFAC levels in plasma

• Cell state characterization:

  • Isolate stem cells from distant tissues using FACS (MET+/PAX7+ for muscle stem cells)

  • Compare G₀ versus GAlert markers

  • Measure response kinetics to secondary stimulation

  • Use HGFAC antibodies to neutralize activity and assess impact on state transition

• Signaling pathway dissection:

  • Implement MET inhibitors to block HGFAC-mediated signaling

  • Analyze cross-talk with complementary alarmin pathways (HMGB1-CXCL12-CXCR4)

  • Measure CXCR4 surface expression with/without MET inhibition

  • Assess combined neutralization of multiple alarmin pathways

• In vivo tissue priming model:

  • Pre-treat animals with neutralizing HGFAC antibodies

  • Create primary injury

  • After defined interval, create secondary injury at distant site

  • Compare regeneration efficiency between HGFAC-neutralized and control animals

• Comparative species analysis:

  • Test HGFAC antibody cross-reactivity between species (human, mouse, rat, monkey)

  • Compare alarmin function conservation across evolutionary distance

  • Assess species-specific differences in HGFAC-mediated stem cell priming

These approaches provide a comprehensive framework for characterizing HGFAC's systemic alarmin function and its potential therapeutic implications for enhancing regenerative capacity.

What confounding factors should researchers address when using HGFAC antibodies in complex disease models?

When employing HGFAC antibodies in complex disease models, researchers must account for several confounding factors that can significantly impact experimental outcomes and interpretations:

• Proteolytic cascade interactions:

  • HGFAC functions within complex proteolytic networks

  • Other proteases (KLK-4, KLK-5) can activate proHGFAC

  • Coagulation cascade activation in disease models can indirectly affect HGFAC

  • Control for these interactions by measuring multiple proteases simultaneously

• Glycosaminoglycan variations:

  • Dextran sulfate, chondroitin sulfate, and heparin significantly enhance HGFAC activation by thrombin

  • Disease states alter tissue glycosaminoglycan composition

  • Measure glycosaminoglycan content in experimental tissues

  • Consider how disease-specific glycosaminoglycan alterations affect HGFAC localization and activity

• Inhibitor compensation mechanisms:

  • Dynamic regulation between multiple HGFAC inhibitors (PCI, HAI-1/SPINT1, HAI-2/SPINT2)

  • Disease states may alter inhibitor expression patterns

  • Complex may form reversible complexes releasing active HGFAC under specific conditions

  • Monitor inhibitor levels and complex formation alongside HGFAC measurements

• Inflammatory context variations:

  • Inflammation significantly impacts HGFAC-mediated proHGF activation

  • Different disease models present distinct inflammatory signatures

  • CCl₄-induced liver injury shows substantial HGFAC-dependent regeneration

  • Partial hepatectomy demonstrates HGFAC-independent regeneration

  • Measure inflammatory markers (IL-1, TNF-α) concurrently with HGFAC analysis

• Antibody cross-reactivity concerns:

  • Structural similarity between HGFAC and factor XII

  • Potential cross-reactivity with related serine proteases

  • Validate antibody specificity in each specific disease model

  • Include appropriate genetic knockout controls when possible

Addressing these confounding factors through careful experimental design and comprehensive analysis will enhance the reliability and interpretability of HGFAC antibody-based studies in complex disease models.

What are common technical challenges when using HGFAC antibodies and how can they be resolved?

Researchers frequently encounter several technical challenges when working with HGFAC antibodies. Here are effective solutions to address these issues:

• Inconsistent western blot detection:

  • Problem: Variable or weak band detection at expected 70 kDa

  • Solution: Optimize protein extraction using freshly prepared RIPA buffer supplemented with protease inhibitors. Ensure complete transfer by using PVDF membrane for higher protein retention. Increase primary antibody concentration (1:500) and extend incubation time to overnight at 4°C .

• Non-specific binding:

  • Problem: Multiple bands or high background

  • Solution: Increase blocking time (2 hours minimum) with 5% BSA instead of milk. Include additional washing steps (5×5 minutes) with 0.1% Tween-20 in TBS. Consider using more stringent washing conditions and titrating the antibody to optimal concentration .

• Cross-reactivity issues:

  • Problem: Unexpected bands from related serine proteases

  • Solution: Validate specificity using immunizing peptide competition assays. Include knockout/knockdown samples as negative controls when available. For applications in non-validated species, perform preliminary cross-reactivity testing with appropriate controls .

• Sample degradation:

  • Problem: Inability to detect intact HGFAC

  • Solution: Add protease inhibitor cocktail immediately during sample collection. Process samples quickly and maintain cold chain. Avoid repeated freeze-thaw cycles (store at -80°C in single-use aliquots) .

• Detecting activated forms:

  • Problem: Difficulty distinguishing proHGFAC from activated HGFAC

  • Solution: Use non-reducing conditions to preserve disulfide bonds between chains of activated HGFAC. Consider using antibodies specific to the activation domain or activation peptide.

When working with liver serum samples specifically, centrifuge samples at high speed (20,000×g for 15 minutes) to remove lipids and particulates that can interfere with antibody binding. Dilute samples appropriately (1:5 to 1:20) to minimize matrix effects .

How can researchers determine whether HGFAC antibodies will work in non-validated species?

Determining cross-species reactivity of HGFAC antibodies requires a systematic approach:

• Sequence homology analysis:

  • Align HGFAC protein sequences across target species

  • Focus particularly on the immunogen region used to generate the antibody

  • Higher homology (>85%) in the immunogen region suggests greater likelihood of cross-reactivity

  • Example: A customer inquired about using anti-HGFAC antibody (originally validated for human, mouse, rat) on monkey tissues

• Preliminary validation protocol:

  • Step 1: Start with western blot using positive control samples from validated species alongside experimental species

  • Step 2: Use tissues known to express high levels of HGFAC (liver is optimal)

  • Step 3: Include multiple antibody dilutions (1:250, 1:500, 1:1000, 1:2000)

  • Step 4: Assess band pattern, intensity, and molecular weight compared to validated species

• Confirmatory approaches:

  • Immunoprecipitation followed by mass spectrometry to confirm target identity

  • Preabsorption with immunizing peptide to verify signal specificity

  • Parallel analysis with antibodies targeting different HGFAC epitopes

  • Functional validation using activity assays specific to HGFAC

• Optimization strategies for cross-species application:

  • Increase antibody concentration (up to 2× recommended)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Modify extraction buffer composition to optimize for target species

  • Consider different detection systems for enhanced sensitivity

How should researchers interpret contradictory results between HGFAC expression and activity levels?

When faced with discrepancies between HGFAC expression and activity measurements, researchers should implement the following analytical framework:

• Distinguish between protein presence and activation state:

  • HGFAC exists primarily as inactive proHGFAC in circulation

  • Western blot detection measures total HGFAC protein regardless of activation state

  • Activity assays measure functional capacity, which requires proper activation

  • Analyze samples using both reducing (total protein) and non-reducing (activation state) conditions

• Consider inhibitor regulation:

  • High HGFAC protein levels with low activity may indicate inhibitor presence

  • Measure PCI, HAI-1/SPINT1, and HAI-2/SPINT2 levels in parallel

  • HAI-1/SPINT1 forms reversible complexes that can be disrupted under certain conditions

  • Test for HGFAC-inhibitor complexes using co-immunoprecipitation

• Evaluate microenvironmental factors:

  • Glycosaminoglycans significantly enhance HGFAC activation

  • Local inflammation alters protease networks

  • Measure tissue glycosaminoglycan content and inflammatory markers

  • In acetic acid-induced and dextran sodium sulfate-induced colitis models, robust proHGF activation occurs despite variable HGFAC expression

• Analyze experimental model specificity:

  • Different injury models show distinct HGFAC activation patterns

  • CCl₄-induced liver injury demonstrates significant HGFAC-dependent proHGF activation

  • Partial hepatectomy shows minimal HGFAC-dependent activation

  • Cross-validate findings across multiple experimental models

• Technical resolution approaches:

  • Employ activity-based protein profiling with active-site directed probes

  • Use conformation-specific antibodies when available

  • Implement parallel methodologies (western blot, zymography, activity assays)

  • Include time-course analyses to capture transient activation events

This systematic approach allows researchers to resolve apparent contradictions between HGFAC expression and activity measurements, leading to more accurate interpretations of experimental results in complex biological systems.