GCHFR Antibody

What is GCHFR and why is it important to study?

GCHFR, also known as GFRP, is an 84 amino acid enzyme inhibitor that binds to and regulates the activity of GTP cyclohydrolase I (GTPCH). GTPCH is the rate-limiting enzyme in the biosynthesis of tetrahydrobiopterin (BH4), an essential cofactor for nitric oxide synthases and aromatic amino acid hydroxylases. GCHFR is typically found as a 20 kDa homodimer in the nucleus and cytoplasm of multiple cell types, including endothelial cells, keratinocytes, and melanocytes .

The study of GCHFR is critical because it helps understand the regulation of BH4 production, which impacts numerous physiological processes including neurotransmitter synthesis, vascular function, and immune responses. Research using GCHFR antibodies allows investigators to elucidate the complex relationship between GCHFR, GTPCH activity, and BH4 production in various disease states.

What GCHFR antibody types are available for research applications?

Several types of GCHFR antibodies are commercially available:

When selecting a GCHFR antibody, researchers should consider the specific reactivity needed (human, mouse, rat), the intended application (Western blot, IHC, ELISA), and whether the experiment requires a conjugated antibody for direct detection or an unconjugated antibody for flexibility in detection methods.

What are the optimal conditions for GCHFR antibody storage and handling?

To maintain GCHFR antibody functionality:

• Store antibodies at -20°C in aliquots to avoid repeated freeze/thaw cycles that can degrade antibody quality .

• For long-term storage (>6 months), most suppliers recommend -70°C storage conditions .

• For reconstituted antibodies, store at 2-8°C for up to 1 month under sterile conditions .

• Most GCHFR antibodies are supplied in buffer solutions containing preservatives like sodium azide and stabilizers like glycerol .

• When handling, avoid contamination and follow supplier recommendations for thawing (typically slow thawing on ice).

Proper storage is essential for maintaining antibody performance, as degraded antibodies can lead to inconsistent results, higher background, and reduced specificity.

How should researchers optimize GCHFR antibody dilutions for different applications?

Optimizing antibody dilutions is critical for balancing signal strength and background. Based on available data:

For Western Blot applications:

• Starting dilution range: 1/500 - 1/2000

• For Prestige Antibodies: 0.04-0.4 μg/mL

• Validation shown with human hepatocellular carcinoma cell lines at 1 μg/mL

For Immunohistochemistry applications:

• Typical range: 1/20 - 1/200

• For Prestige Antibodies: 1:50-1:200

• For fluorescence-conjugated antibodies: Experimental determination recommended

For ELISA applications:

• Starting dilution: 1/10000

Optimal dilutions should be determined experimentally for each specific application, antibody lot, and sample type. Begin with the manufacturer's recommended range and perform a dilution series to identify the concentration that provides the best signal-to-noise ratio for your specific experimental conditions.

What sample preparation methods are most effective for GCHFR detection?

Effective sample preparation is essential for successful GCHFR detection:

For Western Blotting:

• Use reducing conditions as demonstrated in studies with human hepatocellular carcinoma cell lines .

• Standard lysis buffers containing protease inhibitors are suitable.

• Consider using Immunoblot Buffer Group 1 as specified in validation studies .

• Be aware that GCHFR appears at approximately 10-11 kDa on SDS-PAGE gels .

For Immunohistochemistry:

• Formalin-fixed, paraffin-embedded tissues have been successfully used for GCHFR detection.

• Appropriate antigen retrieval methods are critical and may be antibody-specific.

• For fluorescent detection, minimize exposure to light when using conjugated antibodies like DyLight 488 .

For Cell Culture Studies:

• When working with cell lines, consider those validated for GCHFR expression, such as Hep3B, HepG2, and Bowes human melanoma cell lines .

How can GCHFR antibodies be used to investigate the regulatory relationship between GCHFR and GCH1?

GCHFR antibodies are valuable tools for investigating the complex relationship between GCHFR and GCH1. Research methodologies include:

• Co-immunoprecipitation studies: Use GCHFR antibodies to pull down protein complexes and detect GCH1 association, revealing insights into their physical interaction under different conditions.

• Expression correlation analysis: Parallel detection of GCHFR and GCH1 in experimental models can reveal regulatory relationships:

  • Research has demonstrated that despite 50-fold changes in GCH1 expression and BH4 levels, GCHFR expression and protein levels remained unchanged in certain experimental models .

  • These findings suggest that GCH1 expression is the primary determinant of cellular BH4 levels, with a direct linear relationship between GCH1 mRNA expression and steady-state intracellular BH4 levels .

• Knockdown experiments: siRNA-mediated knockdown of GFRP (reducing protein by 84%) allowed researchers to determine that GTPCH activity and BH4 levels were not significantly altered, suggesting complex regulatory mechanisms beyond simple protein-protein interactions .

• Immunolocalization studies: Use immunofluorescence with GCHFR antibodies to determine subcellular localization and potential co-localization with GCH1 under different conditions.

What considerations are important when using GCHFR antibodies in genetic models of BH4 deficiency?

When utilizing GCHFR antibodies in genetic models like the hph-1 mouse (with graded deficiency in GCH1 expression):

• Antibody cross-reactivity validation: Ensure the selected antibody recognizes the species-specific GCHFR (human GCHFR shares 93% amino acid sequence identity with mouse and rat GCHFR) .

• Genetic background considerations:

  • The breeding of heterozygote pairs of hph-1 mice provides an in vivo model of quantitative reduction in GCH1 expression across an approximately 10-fold range on an otherwise identical genetic background .

  • Immunoblotting for mouse GTPCH protein revealed a stepwise reduction from wildtype to heterozygous to homozygous hph littermates, paralleled by quantitative changes in GTPCH enzymatic activity and tissue BH4 levels .

• Multiple detection methods: Combine antibody-based detection with mRNA expression analysis and enzymatic activity assays to comprehensively characterize the model.

• Tissue specificity: Consider that GCHFR regulation may vary between tissues; liver tissue has been successfully used in hph-1 mouse studies .

How do researchers address the challenges of detecting low-abundance GCHFR protein?

GCHFR is a relatively low-abundance protein with a low molecular weight (observed MW: 10-11 kDa), presenting several detection challenges:

• Sample enrichment techniques:

  • Consider subcellular fractionation to concentrate GCHFR from relevant compartments.

  • Use immunoprecipitation to enrich GCHFR before detection.

• Detection optimization for Western blot:

  • Use appropriate separation techniques: Consider higher percentage gels (15-20%) for better resolution of low molecular weight proteins.

  • Transfer optimization: Use specialized transfer conditions for low molecular weight proteins.

  • Signal enhancement: Consider more sensitive detection systems such as chemiluminescent substrates with enhanced sensitivity.

• Validation strategies:

  • Use positive controls: Cell lines with known GCHFR expression (e.g., HepG2, Hep3B) .

  • Include recombinant GCHFR protein as a standard.

  • Employ RNA interference to confirm signal specificity by comparing detection in normal vs. knockdown samples.

• Alternative detection methods:

  • Simple Western™ technology has been validated for GCHFR detection in human hepatocellular carcinoma cell lines .

  • Mass spectrometry-based approaches for orthogonal validation.

What controls should be included when using GCHFR antibodies in research?

Robust experimental design requires appropriate controls:

• Positive controls:

  • Cell lines with confirmed GCHFR expression: HepG2, Hep3B, Bowes human melanoma, and XB2 mouse teratoma keratinocyte cell lines have been validated .

  • Recombinant GCHFR protein as a standard (available as E. coli-derived recombinant human GCHFR, Met1-Glu84) .

• Negative controls:

  • Primary antibody omission control to assess secondary antibody specificity.

  • Isotype control (matched IgG) to evaluate non-specific binding.

  • siRNA knockdown samples (demonstrated to reduce GFRP protein by 84%) .

• Loading controls:

  • For Western blotting, α-Actinin has been validated as a suitable loading control in GCHFR studies .

  • Be aware that GAPDH levels may change in some experimental conditions, as observed in GFRP siRNA experiments .

• Peptide competition:

  • Pre-incubation of the antibody with immunizing peptide can confirm specificity.

  • Several GCHFR antibodies use defined peptide sequences as immunogens, such as "CVLDKLERRGFRVL" or "MPYLLISTQIRMEVGPTMVGDEQSDPELMQHLGASKRRALGNNFYEYYVD" .

How can researchers validate GCHFR antibody specificity across different experimental systems?

Validating antibody specificity is crucial for reliable research outcomes:

• Cross-platform validation:

  • Compare results across multiple techniques (e.g., Western blot, IHC, immunofluorescence).

  • Verify that the observed molecular weight matches the expected size (10-11 kDa) .

• Genetic validation approaches:

  • Use siRNA or CRISPR-Cas9 systems to knock down or knock out GCHFR, confirming signal reduction.

  • Heterologous expression systems: Overexpress tagged GCHFR and confirm detection with both tag-specific and GCHFR-specific antibodies.

• Species-specific considerations:

  • Verify epitope conservation when working across species (human GCHFR shares 93% amino acid sequence identity with mouse and rat GCHFR) .

  • For antibodies with defined epitopes like "CVLDKLERRGFRVL" , perform sequence alignment to confirm conservation in the species of interest.

• Orthogonal techniques:

  • Correlate protein detection with mRNA expression using qPCR.

  • Mass spectrometry validation of immunoprecipitated proteins.

What approaches can address inconsistent results in GCHFR antibody-based experiments?

When encountering variability in GCHFR detection:

• Technical optimization:

  • Re-evaluate antibody dilutions using a broader range than initially tested.

  • Modify incubation conditions (time, temperature, blocking reagents).

  • For Western blotting, optimize transfer conditions for low-molecular-weight proteins.

• Sample preparation refinement:

  • Ensure consistent sample preparation between experiments.

  • Consider that GCHFR is found in both nuclear and cytoplasmic compartments; incomplete extraction may lead to variability .

  • Evaluate fresh vs. frozen samples for potential degradation effects.

• Biological considerations:

  • Time-course experiments may be necessary, as protein expression can vary temporally.

  • Consider cell density and culture conditions that might affect GCHFR expression.

  • In animal models, age, sex, and genetic background can influence results.

• Antibody factors:

  • Lot-to-lot variability: Validate new antibody lots against previous ones.

  • Consider using monoclonal antibodies for more consistent results when reproducibility is a primary concern.

  • Evaluate storage conditions and antibody age as potential sources of variability.

How are GCHFR antibodies being used to study the relationship between BH4 biosynthesis and disease models?

GCHFR antibodies have enabled several important research findings:

• GCHFR regulation in metabolic pathways:

  • Studies have demonstrated that GCH1 mRNA expression directly correlates with the protein level and enzymatic activity of GTPCH along with BH4 production in both cultured cells and in the hph-1 mouse model .

  • Despite marked alterations in GCH1 expression and BH4 levels, GFRP mRNA and protein levels remained unchanged, challenging previous paradigms about GCHFR regulation .

• Quantitative relationship analysis:

  • Researchers have established a striking linear relationship between human GCH1 mRNA expression and resulting steady-state intracellular BH4 levels, suggesting that GCH1 expression is the principal determinant of cellular BH4 levels .

  • GCHFR knockdown experiments demonstrated that despite 84% reduction in GFRP protein, GTPCH activity and BH4 levels were not significantly altered in certain experimental contexts .

• In vivo validation:

  • The levels of GCH1 expression between wildtype, heterozygous, and homozygous hph mice were strongly correlated with BH4 levels, confirming that in mouse liver in vivo, GCH1 expression is the primary regulator of BH4 levels .

What methodological advancements have improved detection specificity for GCHFR in complex samples?

Recent technological improvements have enhanced GCHFR detection:

• Advanced detection platforms:

  • Simple Western™ technology has been validated for GCHFR detection in human hepatocellular carcinoma cell lines, providing automated, reproducible quantification .

  • This approach helps address the challenges of detecting low-molecular-weight proteins like GCHFR (approximately 11 kDa).

• Conjugated antibodies:

  • Directly labeled antibodies such as DyLight 488-conjugated GCHFR monoclonal antibodies provide enhanced sensitivity for immunohistochemistry applications while reducing background issues associated with secondary antibody detection .

• Epitope-specific antibodies:

  • Antibodies targeting specific epitopes like "CVLDKLERRGFRVL" allow for more precise targeting of functional domains within the GCHFR protein.

  • Immunogens encompassing larger portions of the protein (e.g., "MPYLLISTQIRMEVGPTMVGDEQSDPELMQHLGASKRRALGNNFYEYYVD" ) may provide broader epitope recognition.

• Affinity improvements:

  • High-purity antibodies (≥95% by SDS-PAGE) purified by immunogen affinity chromatography offer improved specificity for challenging applications .