GFM1 Antibody

What is GFM1 and why is it important in mitochondrial research?

GFM1 (G elongation factor mitochondrial 1) functions as a GTPase that catalyzes the delivery of peptidyl-tRNA from the ribosomal A site to the P site following peptide bond formation . It plays a crucial role in mitochondrial protein translation, interacting with both the 50S large ribosomal subunit near the L7/L12 stalk and the sarcin-ricin region of 23S rRNA . Mutations in the GFM1 gene have been associated with early-onset mitochondrial diseases, particularly affecting the liver, nervous system, and occasionally the kidneys . The protein consists of 751 amino acids in its canonical form (UniProtKB: Q96RP9) and contains 5 Pfam domains that are essential for its function . Due to its critical role in mitochondrial translation, GFM1 has become an important target for researchers studying oxidative phosphorylation disorders and rare genetic diseases.

What types of GFM1 antibodies are commercially available for research applications?

Currently, researchers have access to several types of GFM1 antibodies including:

• Polyclonal rabbit antibodies targeting human GFM1 (e.g., Atlas Antibodies HPA034765)

• Affinity-isolated antibodies in buffered aqueous glycerol solution (e.g., Sigma-Aldrich Anti-GFM1 HPA034764)

These antibodies are compatible with multiple experimental techniques:

• Immunohistochemistry (IHC): Typically used at dilutions of 1:20-1:50

• Western blotting (WB): Recommended concentrations of 0.04-0.4 μg/mL

• Immunofluorescence (ICC-IF): Used for subcellular localization studies

The antibodies are specifically designed for detecting human GFM1 protein and have undergone validation processes including orthogonal RNAseq verification to ensure specificity .

How should I store and handle GFM1 antibodies to maintain their efficacy?

For optimal performance and longevity of GFM1 antibodies, follow these methodological guidelines:

• Storage temperature: Maintain at -20°C for long-term storage .

• Shipping conditions: Antibodies are typically shipped on wet ice to preserve activity .

• Working solution preparation: When preparing dilutions for experiments, use fresh, sterile buffers.

• Freeze-thaw cycles: Minimize repeated freeze-thaw cycles by aliquoting the antibody upon receipt.

• Storage format: Most commercial GFM1 antibodies are supplied in buffered aqueous glycerol solutions that help maintain stability .

When handling primary antibodies for critical experiments like those examining mitochondrial disease tissues, proper storage is especially important as suboptimal handling can lead to diminished signal intensity and potentially false negative results in techniques like immunohistochemistry and western blotting.

What are the typical application protocols for GFM1 antibodies in immunoblotting?

When using GFM1 antibodies for western blotting applications, researchers should consider the following protocol framework:

• Sample preparation:

  • For tissue samples: Homogenize in appropriate lysis buffer

  • For cell cultures: Lyse with detergent-based buffers containing protease inhibitors

• Immunoblotting protocol:

  • Separate proteins using SDS-PAGE

  • Transfer to appropriate membrane (typically PVDF or nitrocellulose)

  • Block with 5% non-fat milk or BSA in TBS-T

  • Incubate with anti-GFM1 antibody (e.g., Abcam ab176786 or ab171945) at 1:1000 dilution at 4°C overnight

  • Wash with TBS-T

  • Incubate with HRP-conjugated secondary antibody (e.g., goat anti-rabbit, Abcam ab6721) at room temperature for 1 hour

  • Wash thoroughly with TBS-T

  • Detect using chemiluminescent substrate and appropriate imaging system

This protocol has been successfully employed in studies examining GFM1 protein expression in liver and kidney tissues from patients with mitochondrial diseases caused by GFM1 mutations .

How can I use GFM1 antibodies to investigate mitochondrial translation defects in disease models?

Investigating mitochondrial translation defects with GFM1 antibodies requires a comprehensive approach:

• Experimental design for detecting translation defects:

  • Compare GFM1 protein levels between patient and control samples using quantitative western blotting

  • Assess multiple OXPHOS complex components simultaneously using antibody cocktails (e.g., total OXPHOS human WB antibody cocktail, Abcam ab110411)

  • Perform immunofluorescence to examine subcellular localization and potential aggregation of GFM1

• Data interpretation framework:

  • Decreased GFM1 levels may indicate protein instability due to mutations

  • Disrupted assembly of mitochondrial complexes (particularly complexes III and V) often accompanies GFM1 defects

  • Reduced activities of mitochondrial complexes I and IV typically suggest combined OXPHOS deficiency

• Multiple tissue analysis:

  • Different tissues may show variable effects from the same GFM1 mutation

  • In reported cases, NDUFA13 (complex I) expression was decreased in liver but unchanged in kidney tissues

  • ATP5A expression (complex V) typically shows decreased levels in both liver and kidney tissues

This methodological approach allows researchers to correlate genetic findings with functional consequences at the protein and cellular level, providing insights into disease mechanisms.

What are the critical considerations when using GFM1 antibodies for histopathological analysis of patient tissues?

When conducting histopathological analyses with GFM1 antibodies, researchers should consider:

• Tissue preparation protocol:

  • Formalin-fixed, paraffin-embedded (FFPE) tissues require appropriate antigen retrieval

  • Fresh frozen tissues may provide better antigen preservation but more challenging handling

  • Post-mortem interval can significantly affect antibody binding and signal quality

• Controls and validation steps:

  • Include tissues from age-matched controls without mitochondrial disease

  • Use tissues known to express GFM1 at different levels as positive controls

  • Implement negative controls (primary antibody omission) to assess background staining

• Interpretation guidelines for GFM1 staining patterns:

  • Normal tissues show distinctive mitochondrial localization patterns

  • Patient tissues with GFM1 mutations typically display markedly decreased protein expression in affected organs (liver, kidney)

  • Compare staining intensity and pattern across multiple tissue types from the same patient

• Correlation with clinical and genetic data:

  • Patients with GFM1 mutations commonly present with encephalopathy, liver disease, lactic acidosis, and failure to thrive

  • Histological changes often include irregular hepatic plates, steatosis, and cholestasis in liver tissue

  • Glomerular and tubular necrosis may be observed in kidney tissues

This approach has successfully demonstrated diminished GFM1 protein expression in affected tissues from patients with compound heterozygous GFM1 mutations .

How can I differentiate between isoforms of GFM1 protein using antibodies in experimental systems?

Differentiating between GFM1 isoforms requires careful antibody selection and experimental design:

• GFM1 isoform characteristics:

  • Canonical isoform (GFM1-001, ENST00000486715): 751 amino acids (UniProtKB: Q96RP9)

  • Non-canonical isoform 2 (GFM1-004, ENST00000264263): 770 amino acids, includes additional exon between exons 5 and 6 (exon 5b)

• Antibody selection strategy:

  • Verify the exact epitope recognized by the antibody

  • For isoform-specific detection, choose antibodies targeting regions unique to each isoform

  • Consider using antibodies raised against different epitopes to confirm findings

• Experimental validation approach:

  • Combine immunoblotting with RT-PCR to correlate protein and mRNA expression

  • Use recombinant expression systems with tagged isoforms as positive controls

  • Consider immunoprecipitation followed by mass spectrometry for definitive isoform identification

• Methodological considerations for splice variant detection:

  • Some GFM1 mutations can activate cryptic splice sites, resulting in altered isoform expression

  • RT-PCR with primers flanking potential splice regions can detect variant transcripts

  • Sequence analysis of amplified cDNA can reveal insertions (e.g., 57 nucleotides from exon 6 of GFM1-004)

This methodological framework has been used to identify the expression of non-canonical GFM1 isoforms in patients with intronic mutations, such as c.689+908 G>A that induces expression of GFM1-004 rather than the canonical GFM1-001 .

What approaches can resolve contradictory results between different GFM1 antibodies in experimental systems?

When faced with discrepancies between different GFM1 antibodies, consider this systematic troubleshooting approach:

• Antibody validation strategy:

  • Verify antibody specificity using positive and negative control samples

  • Test antibodies in GFM1 knockdown/knockout systems when available

  • Compare results using antibodies targeting different epitopes of GFM1

• Technical considerations:

  • Optimize blocking conditions to reduce non-specific binding

  • Test multiple antibody concentrations and incubation conditions

  • Evaluate different detection systems (chemiluminescence vs. fluorescence)

  • Consider native vs. denaturing conditions if epitope accessibility is an issue

• Sample preparation variables:

  • Different fixation methods may affect epitope recognition

  • Protein extraction protocols can influence conformation and antibody binding

  • Post-translational modifications may mask epitopes in specific conditions

• Data integration framework:

  Parameter	Antibody A	Antibody B	Resolution Approach
  Epitope region	N-terminal	C-terminal	Use both to confirm full-length protein integrity
  Species reactivity	Human-specific	Multi-species	Verify species-specific results with genomic data
  Detection method	Direct	Secondary antibody	Test alternative detection systems
  Validation method	Orthogonal RNAseq	Independent	Compare validation methodologies

• Experimental design to resolve contradictions:

  • Implement orthogonal techniques (mass spectrometry, RNA expression analysis)

  • Design competitive binding experiments with recombinant protein fragments

  • Consider the possibility of tissue-specific isoforms or post-translational modifications

This systematic approach helps researchers distinguish between technical artifacts and biologically meaningful variations in GFM1 detection.

How can GFM1 antibodies be used to investigate mitochondrial disease mechanisms in patient-derived samples?

GFM1 antibodies offer powerful tools for elucidating disease mechanisms in patient samples:

• Multi-technique approach for comprehensive analysis:

  • Immunohistochemistry: Assess tissue distribution and expression levels

  • Western blotting: Quantify protein abundance and detect truncated forms

  • Immunofluorescence: Evaluate subcellular localization and co-localization with other mitochondrial markers

• Clinical-molecular correlation methodology:

  • Compare GFM1 expression between patients with different mutation types

  • Analyze protein expression in relation to clinical severity and survival time

  • Studies have shown intrafamilial variability in disease severity despite identical GFM1 mutations

• Protein functional assessment:

  • Analyze impact on OXPHOS complex assembly using blue native PAGE

  • Measure activities of respiratory chain complexes to correlate with GFM1 expression

  • Investigate potential secondary effects on other mitochondrial translation factors

• Application to prenatal diagnosis:

  • GFM1 antibodies have been used to analyze protein expression in amniotic fluid samples

  • This approach, combined with molecular genetic testing, enables prenatal diagnosis of GFM1-related disorders

  • Early diagnosis allows for better management planning for affected infants

Researchers have successfully applied these approaches to characterize novel GFM1 variants and correlate molecular findings with clinical presentations, as demonstrated in studies of siblings with compound heterozygous GFM1 mutations .

What controls should be included when using GFM1 antibodies to evaluate potential therapeutic interventions?

Rigorous control inclusion is essential when evaluating therapeutic interventions:

• Essential controls for valid interpretation:

  • Biological controls:

    • Age-matched healthy control samples

    • Disease controls (non-GFM1 mitochondrial disorders)

    • Isogenic cell lines differing only in GFM1 status

  • Technical controls:

    • Loading controls (β-actin)

    • Antibody specificity controls (peptide competition)

    • Multiple antibodies targeting different GFM1 epitopes

• Time-course experimental design:

  • Baseline measurements before intervention

  • Multiple time points to capture both immediate and delayed effects

  • Recovery phase analysis after intervention withdrawal

• Dose-response relationship assessment:

  • Multiple concentration/dose levels of therapeutic agent

  • Correlation of GFM1 expression with functional outcomes

  • Identification of minimum effective concentration

• Comprehensive outcome measurement:

  Outcome Parameter	Measurement Technique	Relationship to GFM1
  GFM1 protein levels	Western blotting	Direct measure of intervention effect
  OXPHOS complex assembly	Blue native PAGE	Downstream functional outcome
  Mitochondrial translation	35S-methionine labeling	Process directly regulated by GFM1
  Cellular respiration	Seahorse analysis	Functional consequence of GFM1 activity
  Cell viability	MTT assay	Ultimate cellular outcome

This controlled experimental approach ensures that any observed changes in GFM1 expression or function can be reliably attributed to the therapeutic intervention rather than experimental variables or natural disease progression.

What are the most common technical challenges when using GFM1 antibodies and how can they be addressed?

Researchers frequently encounter several challenges when working with GFM1 antibodies:

• Background signal issues:

  • Challenge: High non-specific background in immunohistochemistry or western blotting

  • Solution: Optimize blocking conditions (5% BSA or milk, overnight at 4°C); increase wash duration and frequency; try alternative blocking agents

• Signal intensity problems:

  • Challenge: Weak or undetectable signal despite confirmed GFM1 expression

  • Solution: Test multiple antibody concentrations; implement signal amplification systems; optimize antigen retrieval for tissue sections; consider alternative extraction buffers for protein isolation

• Isoform detection complications:

  • Challenge: Uncertain detection of specific GFM1 isoforms

  • Solution: Verify antibody epitope in relation to known isoforms; use RT-PCR to confirm isoform expression at mRNA level; consider custom antibodies for isoform-specific regions

• Mitochondrial isolation interference:

  • Challenge: Compromised antibody performance in isolated mitochondrial fractions

  • Solution: Optimize mitochondrial isolation protocol to preserve epitope integrity; avoid harsh detergents; consider mild fixation to stabilize mitochondrial structure

• Cross-reactivity concerns:

  • Challenge: Potential cross-reactivity with other GTP-binding proteins

  • Solution: Validate antibody specificity using knockdown experiments; perform peptide competition assays; use multiple antibodies targeting different epitopes

These technical challenges have been encountered and addressed in research investigating GFM1 mutations in rare mitochondrial disorders, where accurate protein detection is critical for phenotype-genotype correlation studies .

How can researchers optimize immunofluorescence protocols for GFM1 detection in mitochondrial localization studies?

Optimizing immunofluorescence for GFM1 mitochondrial localization requires careful attention to methodological details:

• Sample preparation refinement:

  • Fixation method: 4% paraformaldehyde (10-15 minutes) preserves mitochondrial morphology while maintaining antigen accessibility

  • Permeabilization: Gentle permeabilization with 0.1-0.2% Triton X-100 (5-10 minutes) allows antibody access while preserving mitochondrial structure

  • Cell density: Plate cells at 50-70% confluence to allow clear visualization of mitochondrial networks

• Co-localization strategy:

  • Combine GFM1 antibody with established mitochondrial markers (MitoTracker, TOM20, COX IV)

  • Use spectrally distinct fluorophores to minimize bleed-through

  • Include Z-stack imaging to capture the three-dimensional mitochondrial network

• Signal optimization approach:

  • Antibody dilution: Test range from 1:50 to 1:500 to identify optimal signal-to-noise ratio

  • Incubation conditions: Extended incubation (overnight at 4°C) often improves specific binding

  • Signal amplification: Consider tyramide signal amplification for low-abundance detection

• Image acquisition parameters:

  • Use confocal microscopy for improved resolution of mitochondrial structures

  • Implement deconvolution algorithms to enhance signal clarity

  • Establish consistent exposure settings for comparative analyses between samples

• Quantitative analysis framework:

  • Measure co-localization coefficients (Pearson's, Mander's) for objective assessment

  • Analyze mitochondrial morphology parameters (length, branching, area) in relation to GFM1 signal

  • Implement machine learning approaches for unbiased pattern recognition in complex datasets

This optimized approach has been successfully applied in studies examining the subcellular localization of wild-type and mutant GFM1 proteins, revealing important insights into the pathophysiology of GFM1-related mitochondrial diseases .

What emerging applications of GFM1 antibodies should researchers consider for advancing mitochondrial disease research?

Several innovative applications of GFM1 antibodies show promise for future research:

• Single-cell analysis approaches:

  • Application of GFM1 antibodies in mass cytometry (CyTOF) for single-cell protein profiling

  • Integration with single-cell transcriptomics to correlate GFM1 protein levels with gene expression

  • Spatial proteomics to map GFM1 distribution within mitochondrial subcompartments

• Live-cell imaging innovations:

  • Development of cell-permeable GFM1 antibody fragments for real-time monitoring

  • Combination with optogenetic tools to study dynamic GFM1 interactions

  • FRET-based approaches to investigate GFM1 conformational changes during GTP hydrolysis

• Therapeutic monitoring applications:

  • Quantitative assessment of GFM1 expression as a biomarker for treatment response

  • Development of proximity ligation assays to detect GFM1 interactions with therapeutic targets

  • Non-invasive imaging techniques using labeled GFM1 antibodies for in vivo monitoring

• Precision medicine implementation:

  • Patient-specific GFM1 expression profiling to guide personalized treatment approaches

  • Use of GFM1 antibodies to screen compound libraries for molecules that stabilize mutant protein

  • Integration with CRISPR-based therapies to monitor genetic correction outcomes

These emerging applications build upon the foundation of current GFM1 research, where antibodies have already demonstrated utility in characterizing disease mechanisms related to mutations in the GFM1 gene and their impact on mitochondrial translation and OXPHOS complex assembly .