MEFG1 Antibody

What is MEFG1/GFM1 and how does it function in mitochondria?

MEFG1/GFM1 (Mitochondrial Elongation Factor G1) is a mitochondrial GTPase that plays a crucial role in protein synthesis within mitochondria. It catalyzes the GTP-dependent ribosomal translocation step during translation elongation. During this critical process, the ribosome transitions from the pre-translocational (PRE) to the post-translocational (POST) state as the newly formed A-site-bound peptidyl-tRNA and P-site-bound deacylated tRNA move to the P and E sites, respectively .

MEFG1/GFM1 coordinates the movement of tRNA molecules and mRNA while facilitating conformational changes in the ribosome. This protein does not participate in the disassembly of ribosomes from messenger RNA at the termination of mitochondrial protein biosynthesis . The gene is also known by several synonyms including EFG, EFG1, EFGM, GFM, and hEFG1, and its gene ID is 85476 .

What are the standard applications for MEFG1/GFM1 antibodies in research?

MEFG1/GFM1 antibodies serve multiple experimental purposes in mitochondrial research. According to technical data, these antibodies can be employed in several standard applications:

When selecting application methods, researchers should consider that MEFG1/GFM1 is primarily located in the cytoplasm, specifically in mitochondria . The antibody concentration should be optimized for each specific application and experimental system. For immunofluorescence applications, conjugated antibodies (such as AbBy Fluor® 594) provide direct visualization without requiring secondary antibodies, streamlining the detection process .

How should researchers handle and store MEFG1 antibodies to maintain optimal activity?

Proper handling and storage of MEFG1 antibodies is critical for maintaining their activity and specificity. The antibodies are typically provided in aqueous buffered solutions containing components like TBS (pH 7.4), BSA, preservatives (e.g., Proclin300), and glycerol . For long-term stability:

• Store antibodies at -20°C in a non-frost-free freezer to prevent temperature fluctuations

• Aliquot the antibody solution into multiple small volumes upon receipt to avoid repeated freeze-thaw cycles

• When thawing, allow the antibody to equilibrate to room temperature gradually before use

• Keep the antibody on ice while in use during experiments

• Return to -20°C promptly after use

Multiple freeze-thaw cycles can significantly degrade antibody performance, resulting in decreased sensitivity and increased background. For experiments requiring highly consistent results, researchers should consider preparing single-use aliquots based on their typical experimental requirements .

What species reactivity can researchers expect with commercially available MEFG1/GFM1 antibodies?

When selecting MEFG1/GFM1 antibodies for experiments involving various model organisms, researchers should carefully consider species reactivity profiles. Based on available data, commercially available antibodies demonstrate:

• Confirmed reactivity: Mouse

• Predicted reactivity: Human, Rat, Dog, Cow, Sheep, Pig, Horse

This cross-reactivity profile stems from the conservation of MEFG1/GFM1 sequences across species. The specific antibody described in the technical data (bs-13338R-BF594) was generated using a KLH-conjugated synthetic peptide derived from human GFM1, targeting the region between amino acids 401-500 of the 751-amino acid protein . Researchers working with species not listed should perform preliminary validation experiments before proceeding with full-scale studies.

How can researchers differentiate between MEFG1/GFM1 and other mitochondrial proteins in basic experiments?

Differentiating MEFG1/GFM1 from other mitochondrial proteins requires careful experimental design and controls. Standard approaches include:

• Using properly validated antibodies with confirmed specificity for MEFG1/GFM1

• Employing appropriate controls:

  • Positive control: Tissue/cells known to express MEFG1/GFM1 (e.g., high-energy demanding tissues)

  • Negative control: MEFG1/GFM1 knockout cells or siRNA-treated samples

  • Isotype control: Identical concentration of non-specific IgG from the same host species

• Comparing localization patterns with established mitochondrial markers

• Confirming results using orthogonal methods (e.g., mass spectrometry, RNA expression)

For Western blot applications, researchers should verify that the observed band appears at the expected molecular weight for MEFG1/GFM1 (approximately 83 kDa) to distinguish it from other mitochondrial elongation factors or GTPases .

What strategies should researchers employ to validate MEFG1 antibody specificity in their experimental systems?

Rigorous antibody validation is essential for ensuring reliable research outcomes. For MEFG1/GFM1 antibodies, comprehensive validation should include:

• Genetic approaches:

  • CRISPR/Cas9 knockout of GFM1 gene

  • siRNA or shRNA knockdown with at least 2-3 different sequences

  • Overexpression of tagged MEFG1/GFM1 constructs

• Biochemical validation:

  • Peptide competition assays using the immunizing peptide (region 401-500 for the antibody in search results)

  • Orthogonal detection methods (e.g., mass spectrometry)

  • Testing multiple antibody clones against different epitopes

• Application-specific controls:

  • For IF/ICC: Co-localization with established mitochondrial markers

  • For WB: Molecular weight verification and detection of expression changes upon mitochondrial stress

  • For IP: Mass spectrometry confirmation of pulled-down proteins

• Cross-reactivity assessment:

  • Testing against closely related proteins (e.g., EF-G2mt/GFM2)

  • Species-specific validation when working with non-human models

Researchers should document validation evidence systematically and consider their specific experimental conditions when assessing antibody performance.

How can researchers optimize immunofluorescence protocols for MEFG1/GFM1 detection in different tissue types?

Optimizing immunofluorescence protocols for MEFG1/GFM1 requires methodical adjustment of multiple parameters:

• Fixation optimization:

  • For mitochondrial proteins: 4% paraformaldehyde (10-15 minutes) preserves structure while maintaining epitope accessibility

  • Test multiple fixatives (PFA, methanol, acetone) as different epitopes respond differently

  • Avoid overfixation which can mask epitopes

• Antigen retrieval considerations:

  • Heat-induced epitope retrieval: Test citrate buffer (pH 6.0) vs. EDTA buffer (pH 9.0)

  • Enzymatic retrieval: Consider proteinase K for formalin-fixed tissues

  • Optimize retrieval time (10-30 minutes) and temperature

• Permeabilization optimization:

  • For mitochondrial proteins: 0.1-0.3% Triton X-100 (5-10 minutes)

  • Alternative: 0.1-0.5% saponin for more gentle permeabilization

  • Critical for accessing intramitochondrial epitopes

• Antibody parameters:

  • Dilution testing: Start with 1:50-1:200 range and optimize

  • Incubation time/temperature: 1 hour at room temperature vs. overnight at 4°C

  • Secondary antibody selection: Choose appropriate fluorophores for microscopy setup

• Signal amplification strategies:

  • TSA (tyramide signal amplification) for low-abundance targets

  • Biotin-streptavidin systems for enhanced sensitivity

  • Consider directly conjugated primary antibodies (e.g., AbBy Fluor® 594) to reduce background

For each tissue type, researchers should develop a matrix of conditions and systematically test each parameter while maintaining appropriate controls.

What are the methodological considerations for studying MEFG1/GFM1 mutations and their impact on mitochondrial translation?

Investigating MEFG1/GFM1 mutations requires sophisticated experimental approaches:

• Model system selection:

  • Patient-derived cells for clinical mutations

  • CRISPR/Cas9 engineered cell lines for specific mutations

  • Yeast complementation systems utilizing the MEFG1/GFM1 homolog

  • Mouse models for in vivo studies

• Translation assay methodologies:

  • Mitochondrial protein synthesis assay using 35S-methionine labeling

  • Ribosome profiling specific to mitochondrial ribosomes

  • Polysome profiling to assess translation efficiency

  • SILAC or pulsed SILAC for protein synthesis rate measurement

• GTPase activity assessment:

  • Purified protein GTPase assays with colorimetric detection

  • Real-time GTP hydrolysis assays using fluorescent GTP analogs

  • Structure-function analysis of GTPase domain mutations

• Ribosome interaction studies:

  • Cryo-EM to visualize MEFG1/GFM1-ribosome interactions

  • FRET assays to measure conformational changes during translocation

  • Cross-linking and mass spectrometry to map interaction sites

• Cellular phenotype analysis:

  • Oxygen consumption measurements

  • Mitochondrial membrane potential assessments

  • mtDNA-encoded protein expression analysis

  • Mitochondrial morphology and network analysis

These approaches should be combined with proper controls, including wild-type MEFG1/GFM1 and characterized mutations, to establish causality between mutations and observed phenotypes.

How can researchers design experiments to measure MEFG1/GFM1 protein-protein interactions within the mitochondrial translation machinery?

Studying MEFG1/GFM1 interactions requires specialized approaches to maintain native complex integrity:

• Proximity-based methods:

  • BioID or TurboID: Fusion of biotin ligase to MEFG1/GFM1 to biotinylate proximal proteins

  • APEX2 proximity labeling: Generates electron-dense precipitate for EM visualization

  • Split-GFP complementation to visualize interactions in living cells

• Affinity purification strategies:

  • Tandem affinity purification with mild detergents to preserve interactions

  • GFP-Trap or FLAG-tag pulldowns of MEFG1/GFM1

  • Chemical crosslinking prior to purification to capture transient interactions

  • Quantitative mass spectrometry to identify interaction partners

• Structural biology approaches:

  • Cryo-EM of reconstituted translation complexes

  • Hydrogen/deuterium exchange mass spectrometry to map interaction interfaces

  • FRET-based assays to measure dynamic interactions

• In vitro reconstitution:

  • Purified components to recapitulate translation steps

  • Fluorescently labeled components to track movement and interactions

  • Real-time kinetic measurements of complex formation

• Genetic interaction mapping:

  • Synthetic lethality screens

  • Suppressor screens to identify functional partners

  • CRISPR screening for genetic interactions

When analyzing results, researchers should distinguish between direct and indirect interactions, account for the dynamic nature of translation complexes, and validate key interactions through multiple orthogonal methods.

What methodological approaches can researchers use to study the role of MEFG1/GFM1 in mitochondrial disorders?

Investigating MEFG1/GFM1's role in mitochondrial disorders requires multifaceted approaches:

• Patient sample analysis:

  • Immunohistochemistry with MEFG1/GFM1 antibodies (1:50-200 dilution)

  • Western blot analysis of tissue biopsies (1:300-5000 dilution)

  • Genetic sequencing to identify mutations in GFM1 gene

  • Transcriptomics to assess expression changes

• Cellular models:

  • Patient-derived fibroblasts or induced pluripotent stem cells

  • CRISPR-engineered cell lines with patient-specific mutations

  • Transmitochondrial cybrids to isolate mitochondrial effects

  • Tissue-specific differentiated cells relevant to affected tissues

• Functional assessments:

  • Mitochondrial respiration (Seahorse analyzer)

  • Mitochondrial membrane potential (TMRM, JC-1)

  • ATP production assays

  • ROS production measurement

  • mtDNA-encoded protein synthesis rates

• Therapeutic screening approaches:

  • Small molecule screens targeting translation enhancement

  • Gene therapy approaches using viral vectors

  • Mitochondrial-targeted peptides to modulate activity

  • Metabolic bypass strategies

• Animal models:

  • Conditional knockout mouse models

  • Patient mutation knock-in models

  • Phenotypic analysis of tissues affected in human disease

When conducting these studies, researchers should employ relevant controls including age-matched samples for patient studies, isogenic controls for cellular models, and appropriate statistical methods to account for variability in mitochondrial function assays.

How should researchers troubleshoot inconsistent results when using MEFG1/GFM1 antibodies across different experimental conditions?

Systematic troubleshooting of MEFG1/GFM1 antibody performance requires methodical evaluation:

• Antibody-specific factors:

  • Lot-to-lot variation: Test multiple lots if available

  • Storage conditions: Check for proper storage at -20°C with minimal freeze-thaw cycles

  • Age of antibody: Antibody degradation over time

  • Concentration accuracy: Verify with protein assay if needed

• Sample preparation optimization:

  • Extraction methods: Mitochondrial isolation vs. whole cell lysates

  • Buffer compatibility: Test different lysis buffers (RIPA vs. NP-40 vs. Triton)

  • Protease inhibitors: Ensure complete cocktail is used

  • Sample handling: Minimize time at room temperature

• Protocol modifications:

  • Blocking reagents: Test BSA vs. milk vs. commercial blockers

  • Incubation conditions: Time (1h vs. overnight) and temperature (4°C vs. RT)

  • Washing stringency: Buffer composition and number of washes

  • Signal development time: Optimize for specific application

• Systematic validation:

  • Control samples: Run positive and negative controls in parallel

  • Alternative antibodies: Test antibodies targeting different epitopes

  • Complementary techniques: Verify with non-antibody methods

  • Knockdown/knockout validation: Confirm specificity

• Documentation and standardization:

  • Create detailed protocols capturing all variables

  • Maintain antibody validation records

  • Track performance across experiments

  • Consider antibody validation services for critical applications

When troubleshooting, change only one variable at a time and maintain detailed records of all modifications and outcomes to identify the source of inconsistency.

What experimental design considerations are important when studying MEFG1/GFM1 dynamics during cellular stress responses?

Investigating MEFG1/GFM1 behavior during stress requires careful experimental design:

• Stress model selection and validation:

  • Oxidative stress: H₂O₂, paraquat, rotenone

  • ER stress: tunicamycin, thapsigargin

  • Nutrient stress: glucose deprivation, serum starvation

  • Hypoxia: chemical (CoCl₂) or physical (hypoxic chamber)

  • Validation of stress induction using established markers

• Temporal considerations:

  • Time-course experiments: Early (0-2h), intermediate (2-12h), late (12-48h) responses

  • Synchronize cell populations when possible

  • Include recovery phases after stress removal

  • Consider circadian influences on mitochondrial function

• Quantitative assessment methods:

  • Protein level changes: Western blot (1:300-5000 dilution)

  • Localization changes: Live-cell imaging with fluorescent tags

  • Post-translational modifications: Phosphorylation, ubiquitination

  • Turnover rate: Pulse-chase experiments

• Signaling pathway integration:

  • Inhibitor/activator studies to identify regulatory pathways

  • Genetic approaches (CRISPR, siRNA) to validate key regulators

  • Phosphoproteomics to map stress-induced modifications

  • Protein-protein interaction changes during stress

• Multi-parametric analysis:

  • Correlate MEFG1/GFM1 changes with translation rates

  • Measure associated mitochondrial function parameters

  • Assess cell viability and recovery capacity

  • Monitor mitochondrial network dynamics

Researchers should include appropriate controls for each stress condition and consider cell type-specific responses when interpreting results, as mitochondrial stress responses can vary significantly between different tissues and cell types.

How can researchers accurately quantify changes in MEFG1/GFM1 expression across different experimental conditions?

Accurate quantification of MEFG1/GFM1 requires rigorous methodological approaches:

• Protein quantification methods:

  • Western blot with standardized loading controls

    • Use optimized dilutions (1:300-5000)

    • Include linear standard curves

    • Consider mitochondrial markers (VDAC, COX IV) as normalization controls

  • ELISA development for absolute quantification

  • Mass spectrometry-based quantification

    • Label-free quantification

    • TMT/iTRAQ labeling for multiplexed analysis

    • SILAC for turnover studies

• mRNA quantification approaches:

  • RT-qPCR with validated reference genes

  • Digital droplet PCR for absolute quantification

  • RNA-Seq with appropriate normalization

  • Single-cell analysis for heterogeneity assessment

• Image-based quantification:

  • Immunofluorescence with standardized acquisition parameters

    • Use consistent antibody dilutions (1:50-200)

    • Include fluorescence standards

  • High-content imaging for population analysis

  • Colocalization analysis with mitochondrial markers

  • 3D quantification in tissue samples

• Experimental design considerations:

  • Technical replicates (minimum 3)

  • Biological replicates (minimum 3 independent experiments)

  • Randomization and blinding where possible

  • Inclusion of relevant positive and negative controls

  • Inter-assay calibrators for studies performed over time

• Statistical analysis approaches:

  • Power analysis for sample size determination

  • Appropriate statistical tests based on data distribution

  • Correction for multiple comparisons

  • Effect size calculation in addition to p-values

  • Transparent reporting of outliers and exclusions

When integrating multiple quantification methods, researchers should consider the different sensitivities and dynamic ranges of each technique and interpret results accordingly.

What approaches can researchers use to study the long-term durability of MEFG1 antibody responses in immunization studies?

When studying antibody durability in research contexts, investigators can apply methodologies similar to those used in vaccine research:

• Longitudinal sampling strategies:

  • Strategic timepoint selection (baseline, peak, early decline, long-term)

  • Consistent sampling methodology

  • Sample preservation techniques to maintain antibody integrity

  • Paired sample collection for systemic and mucosal compartments

• Quantitative assessment methods:

  • ELISA for antibody titer measurement

  • Surface plasmon resonance for affinity determination

  • Flow cytometry for cell-associated antibody detection

  • Functional assays to assess neutralizing capacity

• Compartment-specific evaluation:

  • Mucosal antibody assessment in relevant tissues

  • Tissue-resident B cell analysis

  • Memory B cell quantification and characterization

  • Plasma cell niche examination

• Experimental design for durability studies:

  • Include heterologous boosting protocols

  • Extended follow-up periods (up to 17 years has been documented)

  • Age stratification to account for immunosenescence

  • Analysis of factors affecting longevity (adjuvants, delivery systems)

• Correlative studies:

  • Molecular signatures predicting durability

  • Germinal center response correlation

  • Antibody glycosylation and other modifications

  • Correlation with T cell helper responses

The remarkable finding that some vaccine-elicited antibody responses can be detected 6-17 years post-vaccination and rapidly boosted upon re-exposure provides a framework for studying antibody durability . While this research focused on HIV-1 vaccine responses, similar principles could be applied to study the longevity of other antibody responses in research settings.

How can MEFG1/GFM1 antibodies be utilized in multiplex imaging approaches to study mitochondrial translation complexes?

Advanced multiplex imaging of mitochondrial translation complexes requires sophisticated methodological approaches:

• Multiplexed fluorescence imaging strategies:

  • Sequential immunofluorescence with MEFG1/GFM1 antibodies (1:50-200 dilution)

  • Spectral unmixing for closely spaced fluorophores

  • Cyclic immunofluorescence (CycIF) for >10 targets

  • DNA-barcoded antibodies for highly multiplexed imaging

  • Proximity ligation assay (PLA) to detect MEFG1/GFM1 interactions

• Super-resolution approaches:

  • STED microscopy for ~50nm resolution

  • STORM/PALM for single-molecule localization

  • SIM for doubled resolution with lower phototoxicity

  • Expansion microscopy to physically enlarge specimens

  • Correlative light and electron microscopy (CLEM)

• Live-cell imaging considerations:

  • SNAP/CLIP/Halo-tagged MEFG1/GFM1 for dynamic studies

  • Photoactivatable/photoconvertible fluorophores for pulse-chase

  • Split fluorescent proteins to visualize interactions

  • FRET/FLIM to measure protein proximity

  • Lattice light-sheet for reduced phototoxicity

• Image analysis methods:

  • Machine learning for feature extraction

  • 3D reconstruction of translation complexes

  • Single-particle tracking for dynamics

  • Colocalization analysis with statistical validation

  • Spatial statistics to quantify clustering

• Mitochondria-specific considerations:

  • Combining with mitochondrial network analysis

  • Correlating with functional readouts (membrane potential)

  • Sub-mitochondrial localization analysis

  • Integration with mitochondrial calcium or ROS imaging

  • Mitochondrial isolation and in vitro reconstitution imaging

When implementing these approaches, researchers should carefully validate all antibodies in multiplex settings, as fixation and permeabilization requirements may differ between targets, and optimize signal-to-noise ratios for each channel to ensure accurate quantification and colocalization assessment.