HGF (32-285) Human

What is HGF (32-285) Human recombinant protein?

HGF (32-285) Human is a recombinant protein fragment containing amino acids 32-285 of the full-length human Hepatocyte Growth Factor. It is typically produced in Escherichia coli as a single, non-glycosylated polypeptide chain with a molecular mass of approximately 29.8 kDa . This protein represents a functional domain of HGF that maintains many of the biological activities of the complete protein while offering advantages in terms of production and experimental manipulation. The protein is also known by several synonyms including Scatter Factor (SF), Hepatopoietin (HPTA), HGFB, F-TCF, and DFNB39 .

What are the key biological functions of HGF (32-285) in research applications?

HGF (32-285) functions as a potent mitogen for mature parenchymal hepatocyte cells and acts as a growth factor for a broad spectrum of tissues and cell types . In research contexts, it serves as an important tool for studying cellular processes including proliferation, motility, and differentiation. HGF (32-285) has been demonstrated to exert significant effects on human mesenchymal stem cells (hMSCs), where it promotes osteogenic differentiation through the transcription of key markers such as osteocalcin, osterix, and osteoprotegerin . Additionally, it synergizes with Interleukin-3 and GM-CSF to stimulate colony formation of hematopoietic progenitor cells in vitro, making it valuable for hematopoiesis research .

How does the amino acid sequence of HGF (32-285) compare to full-length HGF?

The HGF (32-285) fragment contains 255 amino acids with the sequence: MQRKRRNTIH EFKKSAKTTL IKIDPALKIK TKKVNTADQC ANRCTRNKGL PFTCKAFVFD KARKQCLWFP FNSMSSGVKK EFGHEFDLYE NKDYIRNCII GKGRSYKGTV SITKSGIKCQ PWSSMIPHEH SYRGKDLQEN YCRNPRGEEG GPWCFTSNPE VRYEVCDIPQ CSEVECMTCN GESYRGLMDH TESGKICQRW DHQTPHRHKF LPERYPDKGF DDNYCRNPDG QPRPWCYTLD PHTRWEYCAI KTCET . This segment represents the N-terminal portion of the complete HGF protein (P14210), which contains important domains for receptor binding and biological activity.

What are the optimal storage conditions for HGF (32-285) Human?

For optimal stability and activity maintenance, HGF (32-285) should be stored at 4°C if the entire vial will be used within 2-4 weeks . For longer periods, the protein should be stored frozen at -20°C. To minimize activity loss during long-term storage, it is recommended to add a carrier protein such as 0.1% Human Serum Albumin (HSA) or Bovine Serum Albumin (BSA) . Multiple freeze-thaw cycles should be strictly avoided as they can lead to significant degradation of protein structure and function. For research requiring consistent protein activity over extended periods, it is advisable to prepare aliquots upon initial thawing to minimize freeze-thaw events.

How should HGF (32-285) be prepared for cell culture experiments?

HGF (32-285) is typically formulated as a protein solution (1mg/ml) containing 20mM Tris-HCl buffer (pH 8.0), 10% glycerol, and 0.4M Urea . For cell culture applications, the protein should be diluted to the appropriate working concentration in sterile cell culture medium immediately before use. Based on experimental protocols from the literature, an optimal working concentration of 40 ng/mL has been established for many cell-based assays, particularly when working with human mesenchymal stem cells . When planning experiments, consider performing a dose-response assessment (e.g., 10, 20, 40, and 100 ng/mL) to determine the optimal concentration for your specific cell type and experimental endpoint.

What methods can be used to verify HGF (32-285) activity in experimental systems?

Several approaches can be employed to confirm the biological activity of HGF (32-285):

• Phosphorylation assays: HGF activates multiple signaling pathways including rapid phosphorylation of p38, ERK, and AKT/PI3K. Western blot analysis using phospho-specific antibodies can detect these activation events within minutes of treatment .

• Proliferation assays: HGF (32-285) promotes cell proliferation in responsive cell types. A simple hemocytometer-based cell counting method after 5 days of treatment can confirm activity .

• Differentiation markers: In mesenchymal stem cells, HGF (32-285) increases expression of osteogenic markers. RT-qPCR analysis of genes such as osteocalcin, osterix, and osteoprotegerin can validate functional activity .

• Functional blocking: Using HGF-neutralizing antibodies (e.g., MAB294 at 100-300 ng/mL) or c-Met inhibitors (e.g., PHA665752) alongside HGF treatment provides important controls to confirm specificity of observed effects .

What experimental approaches can resolve contradictory results when using HGF (32-285) in different cell types?

When encountering contradictory results across different cell types, several systematic approaches can help resolve discrepancies:

• Receptor expression profiling: Quantify c-Met receptor expression levels across cell types using flow cytometry or Western blotting, as differential receptor densities can significantly impact response magnitude.

• Pathway analysis: Implement comprehensive phosphoproteomic analysis to map activated signaling networks in different cell types, as downstream pathway availability varies between cellular contexts.

• Temporal dynamics assessment: Conduct time-course experiments (0-72 hours) measuring both immediate (signaling) and delayed (transcriptional) responses, as timing differences often explain apparent contradictions.

• Co-factor identification: Analyze culture medium composition for growth factors that might synergize with or antagonize HGF signaling, particularly when using serum-containing media which contains variable growth factor concentrations.

• Genetic background characterization: Sequence key components of the HGF signaling pathway to identify polymorphisms that might affect response characteristics between donor-derived primary cells.

How can researchers distinguish between direct and indirect effects of HGF (32-285) in complex tissue models?

Distinguishing direct from indirect effects in complex models requires sophisticated experimental designs:

• Cell-specific receptor knockout: Implement conditional c-Met receptor deletion in specific cell populations using CRISPR/Cas9 or siRNA approaches.

• Conditioned media experiments: Compare direct HGF treatment with conditioned media from HGF-treated cells to identify secondary paracrine mediators.

• Single-cell transcriptomics: Apply scRNA-seq to identify cell type-specific responses within heterogeneous populations, revealing which cells respond directly versus those affected through paracrine mechanisms.

• Pharmacological pathway dissection: Use selective inhibitors of potential mediator pathways in combination with HGF treatment to block indirect effects while preserving direct signaling.

• Temporal segregation: Implement pulse-chase experiments with labeled HGF followed by washout periods to separate immediate direct effects from delayed indirect responses.

What controls are essential when designing experiments with HGF (32-285)?

A robust experimental design with HGF (32-285) should include the following controls:

How should researchers approach HGF (32-285) concentration optimization for different experimental endpoints?

Concentration optimization should be approached systematically based on the experimental endpoint:

For immediate signaling events (0-60 minutes), begin with a range of 10-100 ng/mL HGF and measure phosphorylation of key pathway components (p38, ERK, AKT) using Western blotting or ELISA-based methods. The optimal concentration may differ between pathways, and this information should guide subsequent experiments targeting specific mechanisms.

For transcriptional responses (6-48 hours), initial testing should include concentrations from 10-40 ng/mL, with gene expression analysis performed using RT-qPCR for known HGF-responsive genes. Published data indicates that 40 ng/mL is often optimal for transcriptional effects in mesenchymal stem cells .

For long-term phenotypic changes such as differentiation or mineralization, which may require repeated treatments over days to weeks, lower concentrations (20-40 ng/mL) are typically more appropriate to avoid receptor desensitization. Medium changes containing fresh HGF should be performed every 3-4 days to maintain consistent signaling .

What methodological approaches can minimize variability when working with HGF (32-285) in primary cell cultures?

Primary cell cultures present inherent variability that can be minimized through several methodological approaches:

• Donor pooling: Using pooled cells from multiple donors can reduce donor-specific variability while maintaining primary cell characteristics.

• Passage standardization: Limit experiments to specific passage numbers (typically passages 3-7) to avoid senescence-related changes in receptor expression.

• Receptor quantification: Implement flow cytometry-based quantification of c-Met receptor expression as a pre-experiment quality control measure.

• Serum standardization: Either use serum-free conditions or implement batch-testing of serum lots to select those with minimal impact on baseline HGF signaling.

• Synchronized cell cycle: Implement serum starvation (6-12 hours) prior to HGF treatment to synchronize cells in G0/G1, reducing cell cycle-dependent variability in responses.

• Internal normalization: Include housekeeping genes or proteins that remain stable during HGF treatment as internal controls for quantitative analyses.

How does HGF (32-285) compare functionally to full-length HGF and other growth factors in stem cell applications?

The functional comparison between HGF variants and other growth factors reveals distinct biological profiles:

HGF (32-285) maintains many of the biological activities of full-length HGF while offering advantages in terms of production efficiency and stability. While it effectively activates the p38 MAPK pathway critical for osteogenic differentiation , its effects on STAT3 signaling may be attenuated compared to the full-length protein. This makes HGF (32-285) particularly suited for research focused on osteogenic differentiation and migration, while full-length HGF may be preferred for studies requiring the complete spectrum of biological activities.

What biological effects of HGF (32-285) have been validated across multiple experimental systems?

Several key biological effects of HGF (32-285) have been consistently observed across diverse experimental systems:

• Promotion of cell migration: HGF (32-285) enhances cellular motility in various cell types, including mesenchymal stem cells, epithelial cells, and certain cancer cell lines.

• Activation of p38 MAPK signaling: Rapid phosphorylation of p38 MAPK, particularly the α and β isoforms, has been documented in multiple cell types following HGF (32-285) treatment .

• Osteogenic differentiation: HGF (32-285) consistently promotes osteogenic differentiation of mesenchymal stem cells, as evidenced by increased expression of osteocalcin, osterix, and osteoprotegerin .

• Synergy with hematopoietic factors: HGF (32-285) synergizes with Interleukin-3 and GM-CSF to enhance colony formation of hematopoietic progenitor cells .

• c-Met receptor dependent signaling: The biological effects of HGF (32-285) are consistently abolished by c-Met inhibitors or receptor knockdown, confirming the specificity of its mechanism across systems .

These consistent observations provide a solid foundation for researchers to build upon when designing experiments in novel cell types or experimental conditions.

What are common challenges when using HGF (32-285) in long-term differentiation studies and how can they be addressed?

Long-term differentiation studies with HGF (32-285) present several challenges that require specific methodological solutions:

• Protein stability: HGF (32-285) may degrade during extended culture periods. Solution: Implement a regular supplementation schedule with fresh protein every 3-4 days rather than relying on a single initial treatment .

• Receptor desensitization: Continuous exposure to high concentrations can downregulate c-Met receptor expression. Solution: Use moderate concentrations (20-40 ng/mL) and consider implementing pulsed treatment protocols with recovery periods.

• Serum interactions: Components in serum may interact with HGF signaling over time. Solution: Consider defined serum-free media formulations for critical experiments, particularly when comparing conditions.

• Paracrine signaling changes: Long-term HGF treatment alters the secretory profile of treated cells. Solution: Implement regular medium changes to prevent accumulation of secondary mediators that might confound results.

• Heterogeneous responses: Extended differentiation protocols can lead to population heterogeneity. Solution: Implement single-cell analysis techniques or cell sorting to identify and characterize responder subpopulations.

How can researchers effectively troubleshoot unexpected experimental outcomes with HGF (32-285)?

When encountering unexpected results with HGF (32-285), implementing a systematic troubleshooting approach can identify underlying issues:

• Verify protein activity: Conduct a simple phospho-p38 Western blot in a known responsive cell line to confirm the activity of your HGF (32-285) preparation.

• Assess receptor expression: Quantify c-Met receptor levels in your experimental cells using flow cytometry or Western blotting, as expression levels vary widely between cell types and culture conditions.

• Examine buffer compatibility: The Tris-HCl and urea components in HGF formulation may interact with certain media components. Test dilution in different buffers before adding to cells.

• Evaluate batch-to-batch variability: If using a new lot of HGF (32-285), run side-by-side comparisons with previous lots on a standardized assay to identify potential activity differences.

• Check for inhibitory factors: Some cell types secrete HGF antagonists or express truncated c-Met receptors that can act as decoys. Analyze culture supernatants for potential interfering molecules.

• Consider timing dynamics: HGF responses follow complex temporal patterns. Implement a detailed time-course analysis (minutes to days) to ensure you're not missing the relevant window for your endpoint.

• Examine experimental conditions: Cell density, attachment substrate, and oxygen tension can all significantly impact HGF responsiveness. Systematically vary these parameters to identify optimal conditions for your system.

What advanced analytical techniques are most informative for studying HGF (32-285) molecular mechanisms?

Advanced analytical approaches provide deeper insights into HGF (32-285) mechanisms:

• Phosphoproteomics: Mass spectrometry-based phosphoproteomic analysis can comprehensively map signaling networks activated by HGF beyond the canonical pathways, revealing unexpected connections and feedback mechanisms.

• ChIP-seq: Chromatin immunoprecipitation followed by sequencing identifies the genome-wide binding patterns of transcription factors activated downstream of HGF signaling, revealing the complete transcriptional program.

• Proximity labeling: BioID or APEX2-based proximity labeling coupled with mass spectrometry can identify proteins that dynamically interact with c-Met receptor following HGF binding.

• Live-cell biosensors: FRET-based biosensors for key kinases (p38, ERK) allow real-time visualization of signaling dynamics in living cells with subcellular resolution.

• Ribosome profiling: Provides genome-wide information on actively translated mRNAs following HGF treatment, distinguishing between transcriptional and translational regulation.

• Spatial transcriptomics: In complex tissue models, spatial transcriptomics can map HGF-induced gene expression changes with preserved spatial information, revealing intercellular communication networks.