GFER Antibody

What is GFER and why is it an important research target?

GFER, also known as Augmenter of Liver Regeneration (ALR), is an approximately 30 kDa mitochondrial sulfhydryl oxidase that plays critical roles in multiple cellular processes . In humans, the canonical protein consists of 205 amino acid residues with a molecular mass of 23.4 kDa . GFER is ubiquitously expressed across many tissue types and has multiple subcellular localizations including the mitochondrial intermembrane space, cytoplasm, and is also secreted .

The protein is involved in cellular responses to lipopolysaccharides and TNF-mediated signaling pathways . GFER's significance as a research target stems from its critical roles in liver regeneration, mitochondrial function, and hematopoietic stem cell regulation. Mutations in GFER have been associated with mitochondrial myopathy, highlighting its clinical relevance .

What applications are most commonly used with GFER antibodies?

GFER antibodies have been validated for multiple experimental applications, with Western blotting (WB) being the most widely utilized technique . Other common applications include:

• Immunohistochemistry (IHC) - For detection of GFER in tissue sections, with validated protocols for both paraffin-embedded and frozen tissues

• Enzyme-Linked Immunosorbent Assay (ELISA) - For quantitative detection of GFER in solution

• Immunocytochemistry (ICC) - For subcellular localization studies in cultured cells

• Flow cytometry (FACS) - For quantitative analysis of GFER expression in cell populations

When designing experiments, researchers should consider that GFER antibodies have been successfully used to detect the protein in various human tissues, with specific validation in liver samples and hepatocellular carcinoma cell lines such as HepG2 and Hep3B .

How should I optimize Western blot protocols for GFER detection?

For optimal Western blot detection of GFER, consider the following methodological approach:

• Sample preparation: Use reducing conditions as demonstrated in validated protocols

• Gel selection: Since GFER has a molecular weight of approximately 20-24 kDa, 12-15% SDS-PAGE gels are recommended for optimal resolution

• Transfer: PVDF membranes are preferred over nitrocellulose for GFER detection

• Antibody concentration: Start with 2 μg/mL for monoclonal antibodies as a baseline and adjust as needed

• Secondary antibody: HRP-conjugated secondary antibodies work well with established detection systems

In HepG2 and Hep3B human hepatocellular carcinoma cell lines, GFER typically appears as specific bands at approximately 20-24 kDa when analyzed under reducing conditions using Immunoblot Buffer Group 1 . If multiple bands appear, this may represent different isoforms, as up to two different isoforms have been reported for this protein .

What controls should I include when working with GFER antibodies?

When designing experiments using GFER antibodies, include the following controls:

• Positive tissue/cell controls:

  • Human liver tissue for immunohistochemistry

  • HepG2 and Hep3B cell lysates for Western blotting

  • For gene expression studies, human tissues from the Human Total Master Panel II have been validated

• Loading/housekeeping controls:

  • GAPDH has been validated as a housekeeping gene for qPCR normalization in GFER mRNA quantification

  • For Western blotting, standard loading controls such as β-actin or GAPDH are appropriate

• Negative controls:

  • Primary antibody omission

  • Non-specific IgG of the same species and isotype as the GFER antibody

• Recombinant protein controls:

  • E. coli-derived recombinant human GFER/ALR (Arg82-Asp205) has been used as a positive control in antibody validation

How does GFER regulate hematopoietic stem cell (HSC) function and quiescence?

GFER plays a critical role in regulating HSC proliferation and maintaining stem cell quiescence through a complex molecular mechanism:

• GFER promotes quiescence and maintains the functional integrity of HSCs by restricting unwarranted proliferation

• Mechanistically, GFER binds to the COP9 signalosome subunit Jab1, preventing its association with p27^kip1^

• This interaction leads to stabilization and nuclear retention of p27^kip1^, a key cell cycle inhibitor

• Knockdown (KD) of Gfer in cKit+Sca1+Flk2-CD34-Lineage- (KLS) cells results in:

  • Loss of cell quiescence

  • Compromised ability to engraft in the bone marrow of lethally irradiated recipient mice

  • Significant reduction in p27^kip1^ levels

• Conversely, overexpression of Gfer elevates the level and nuclear retention of p27^kip1^

This Gfer-mediated pro-quiescence mechanism could potentially be therapeutically exploited in the treatment of hematological malignancies associated with elevated Jab1 and reduced p27^kip1^ levels .

What methods are most effective for studying GFER mutations in relation to mitochondrial diseases?

When investigating GFER mutations in relation to mitochondrial diseases, researchers should employ a multifaceted methodological approach:

• Genetic analysis:

  • Retrotranscribe wild-type GFER cDNA from control muscle tissue for comparative studies

  • Clone the cDNA into expression vectors (e.g., pAcGFP1-N2) for functional studies

  • Propagate recombinant vectors in ultracompetent E. coli cells for stability

• Cell-based functional studies:

  • Use patient and control primary fibroblasts for transfection experiments

  • Consider electroporation with the Human Dermal Fibroblast kit for efficient transfection

  • Establish stable cell lines (e.g., HEK293) expressing wild-type and mutated GFER cDNA for comparative studies

  • Evaluate transfection rates and expression using fluorescence microscopy and FACS analysis

• mRNA quantification:

  • Reverse transcribe total RNA and quantify by qPCR using the ΔΔCt method

  • Use appropriate gene expression assays (e.g., Taqman Hs00193365_m1 for GFER)

  • Include GAPDH as a control housekeeping gene (Hs99999905_m1)

  • Compare expression in patient tissues with control human tissues

• Mitochondrial function assessment:

  • Evaluate complex IV activity in GFER mutants

  • Assess genetic stability of mtDNA

  • Examine mitochondrial morphological changes using appropriate imaging techniques

These methods have successfully established GFER's role in the human Disulfide Relay System (DRS) and enhanced understanding of the pathogenesis of a novel mitochondrial disease .

What are the key considerations for selecting the optimal GFER antibody for specific research applications?

Selecting the optimal GFER antibody requires careful consideration of several factors based on your specific research application:

Key selection considerations include:

• Target region specificity:

  • For full-length GFER detection, antibodies targeting amino acids 1-198 are available

  • For specific isoform detection, choose antibodies targeting unique regions (e.g., C-terminal epitopes)

  • For functional studies, consider antibodies targeting the functional domains (e.g., AA 81-205)

• Host species and cross-reactivity:

  • Rabbit polyclonal antibodies typically offer reactivity across human, mouse, and rat samples

  • Mouse monoclonal antibodies may offer higher specificity but potentially more limited cross-reactivity

• Validation evidence:

  • Prioritize antibodies with documented validation in your specific application and cell/tissue type

  • Consider antibodies with multiple validation methods (WB, IHC, ICC)

• Buffer and storage requirements:

  • Most GFER antibodies are supplied in phosphate buffered solutions (pH 7.4) with glycerol and require -20°C storage

  • Avoid repeated freeze/thaw cycles to maintain antibody performance

How can I troubleshoot common issues when using GFER antibodies for mitochondrial localization studies?

When conducting mitochondrial localization studies with GFER antibodies, researchers may encounter several challenges. Here are methodological approaches to troubleshoot common issues:

• Poor mitochondrial signal specificity:

  • Use heat-induced epitope retrieval with Antigen Retrieval Reagent-Basic before antibody incubation

  • Perform overnight incubation at 4°C to enhance specific binding

  • Co-stain with established mitochondrial markers to confirm localization

  • Use super-resolution microscopy techniques for better resolution of mitochondrial structures

• High background in immunohistochemistry:

  • Optimize blocking conditions (5% BSA or serum from the same species as the secondary antibody)

  • Reduce primary antibody concentration (start with 15 μg/mL for monoclonal antibodies)

  • Use chromogenic detection systems optimized for mitochondrial proteins (e.g., HRP-DAB Cell & Tissue Staining Kit)

  • Counter-stain with hematoxylin for better visualization of cellular structures

• Multiple bands in Western blot:

  • Verify if bands represent different isoforms (GFER has up to two reported isoforms)

  • Use reducing conditions to eliminate non-specific disulfide interactions

  • Increase washing stringency to reduce non-specific binding

  • Consider using more specific monoclonal antibodies if polyclonal antibodies show excessive cross-reactivity

• Inconsistent results across different cell lines:

  • Be aware that GFER expression varies across tissue types

  • Validate antibody performance in each new cell line before conducting full experiments

  • Use positive control cell lines like HepG2 or Hep3B where GFER detection has been validated

What methodologies can be used to study GFER's role in the mitochondrial disulfide relay system?

Investigating GFER's role in the mitochondrial disulfide relay system (DRS) requires specialized techniques targeting protein-protein interactions and redox biochemistry:

• Protein-protein interaction studies:

  • Identify interaction partners such as Jab1 using co-immunoprecipitation techniques

  • Use fluorescence resonance energy transfer (FRET) to visualize interactions in live cells

  • Apply proximity ligation assays to detect and quantify protein associations in fixed cells

• Functional rescue experiments:

  • Transfect wild-type GFER cDNA into cells with GFER mutations or knockdown

  • Evaluate mitochondrial function recovery through:

    • Complex IV activity measurements

    • mtDNA stability assessments

    • Mitochondrial morphology analysis

• Redox state analysis:

  • Employ redox proteomics to identify substrates of GFER's sulfhydryl oxidase activity

  • Use redox-sensitive fluorescent proteins to monitor real-time changes in mitochondrial redox state

  • Apply mass spectrometry to identify oxidation states of GFER substrates

• Genetic manipulation approaches:

  • Create stable cell lines overexpressing wild-type or mutated GFER for comparative studies

  • Use CRISPR-Cas9 to generate GFER knockout models

  • Apply inducible expression systems to study temporal effects of GFER activity

• Mitochondrial function assessment:

  • Measure oxidative phosphorylation efficiency

  • Assess maintenance of mitochondrial genomes

  • Evaluate effects on the cell division cycle

These methodologies have successfully established that mutations in GFER can reproduce complex IV activity defects, cause genetic instability of mtDNA, and lead to mitochondrial morphological defects, contributing to our understanding of novel mitochondrial diseases .