ATP5MG Antibody

What is ATP5MG and why is it important in mitochondrial research?

ATP5MG (ATP synthase membrane subunit g) is part of the mitochondrial membrane ATP synthase (F1F0 ATP synthase or Complex V) that produces ATP from ADP in the presence of a proton gradient across the inner mitochondrial membrane. This gradient is generated by electron transport complexes of the respiratory chain. ATP5MG functions specifically as a minor subunit located with subunit a in the membrane portion of the F0 domain .

F-type ATPases consist of two structural domains: F1 (containing the extramembraneous catalytic core) and F0 (containing the membrane proton channel), linked together by central and peripheral stalks. During ATP synthesis, the catalytic domain of F1 is coupled via a rotary mechanism of the central stalk subunits to proton translocation . Understanding ATP5MG's role is crucial for research into mitochondrial function, bioenergetics, and related disorders.

What antibody options are available for ATP5MG detection?

Based on the search results, researchers have access to multiple antibody types for ATP5MG detection:

When selecting an antibody, researchers should consider the specific experimental application, required species reactivity, and whether polyclonal variability or monoclonal specificity better suits their research needs .

What is the molecular profile of the ATP5MG protein?

In humans, ATP5MG has the following characteristics:

• Molecular weight: Approximately 11.4 kDa

• Amino acid length: 103 residues

• Subcellular localization: Mitochondria (inner membrane)

• Gene location: Chromosome 11q23.3

• Exon count: 4

• Protein family: ATPase g subunit family

• Alternative names: ATP5L, ATP5JG, ATPase subunit g

The protein is widely expressed across many tissue types, making it relevant for diverse physiological studies . The gene can undergo alternative splicing, resulting in multiple transcript variants that may be detected by certain antibodies .

What are the optimal applications for ATP5MG antibodies?

ATP5MG antibodies have been validated for several experimental applications:

Western blotting appears to be the most widely validated application across different antibody products. Researchers should always perform preliminary experiments to determine optimal conditions for their specific samples and protocols .

How should I validate a new ATP5MG antibody before using it in critical experiments?

Proper validation is essential for ensuring reliable results with ATP5MG antibodies:

• Verify the antibody detects a band of the expected molecular weight (~11 kDa) in Western blot

• Use positive control samples known to express ATP5MG (mitochondria-rich tissues/cells)

• Include negative controls (tissues with minimal expression or knockdown/knockout samples)

• Compare results with published literature or alternative antibodies targeting different epitopes

• Confirm subcellular localization matches expected mitochondrial pattern in IF/IHC

• Check cross-reactivity with other ATP synthase subunits, particularly in complex samples

• Validate reactivity in your specific experimental model (human, mouse, rat, etc.)

For advanced validation, consider using CRISPR knockout or siRNA knockdown approaches to confirm specificity. Recent methodologies for antibody validation described in the literature include high-throughput sequencing and computational analysis to assess binding specificity profiles .

What methodological approaches can optimize Western blot protocols for ATP5MG detection?

Due to the small size of ATP5MG (~11 kDa), special considerations for Western blot detection include:

• Use higher percentage gels (15% SDS-PAGE) for optimal separation of low molecular weight proteins

• Consider gradient gels (4-20%) to better resolve ATP5MG from other mitochondrial proteins

• Optimize transfer conditions for small proteins (shorter transfer times or specialized buffers)

• Test different membrane types (PVDF may retain small proteins better than nitrocellulose)

• For primary antibody incubation, 1:1000 dilution at 4°C overnight is commonly effective

• Include appropriate molecular weight markers that cover the low molecular weight range

• Consider using specialized detection systems optimized for low abundance proteins

• Verify that sample preparation methods effectively solubilize membrane proteins

Researchers should also be mindful that mitochondrial protein detection can be challenging due to their hydrophobic nature and potential post-translational modifications.

How can ATP5MG antibodies be used to investigate mitochondrial complex assembly?

ATP5MG antibodies can provide valuable insights into ATP synthase complex assembly:

• Blue Native PAGE combined with immunoblotting can reveal ATP5MG's integration into intact ATP synthase complexes

• Co-immunoprecipitation using ATP5MG antibodies can identify interaction partners within the complex

• Super-resolution microscopy with labeled ATP5MG antibodies can visualize its spatial organization within mitochondria

• Comparative analysis of ATP5MG levels between normal and disease states can reveal assembly defects

• Pulse-chase experiments with ATP5MG detection can track the kinetics of complex assembly

• Cross-linking mass spectrometry with immunoprecipitation can map protein-protein interactions

• CRISPR/Cas9-mediated knockout followed by rescue experiments can determine ATP5MG's role in assembly

Research has shown that defects in ATP synthase subunits can lead to severe multi-systemic disorders, highlighting the importance of understanding complex assembly mechanisms .

What are the methodological considerations when using ATP5MG antibodies in mitochondrial disease research?

When investigating mitochondrial diseases using ATP5MG antibodies:

• Compare ATP5MG protein levels between patient and control samples using quantitative Western blotting

• Analyze ATP5MG incorporation into ATP synthase complexes using blue native electrophoresis

• Assess mitochondrial morphology and ATP5MG localization using immunofluorescence microscopy

• Consider how genetic variants might alter epitope accessibility or expression levels

• Use multiple antibodies targeting different epitopes to confirm findings

• Correlate ATP5MG findings with functional assays of ATP synthase activity

• Examine tissue-specific differences in ATP5MG expression and complex assembly

• Consider post-translational modifications that might be altered in disease states

Research has demonstrated that mutations in ATP synthase subunits can cause severe infantile multi-systemic disorders with features including hypotonia, developmental delay, cardiomyopathy, and progressive epileptic encephalopathy .

How can advanced computational approaches improve ATP5MG antibody specificity?

Recent research has explored sophisticated methods to enhance antibody specificity:

• Phage display experiments with antibody libraries can select for variants with high specificity to ATP5MG

• High-throughput sequencing and computational analysis can identify optimal binding sequences

• Biophysics-informed modeling can disentangle different binding modes associated with specific ligands

• Machine learning approaches can predict binding properties beyond experimentally observed sequences

• Computational design can generate antibodies with customized specificity profiles

• Integration of experimental selection with in silico analysis can mitigate biases in antibody development

• Structure-based computational approaches can optimize antibody-antigen interactions

This methodology involves identifying different binding modes associated with particular ligands against which the antibodies are selected. These advanced approaches can be particularly valuable when trying to discriminate between structurally and chemically similar ligands .

What are common issues when working with ATP5MG antibodies and how can they be resolved?

Researchers may encounter several challenges when working with ATP5MG antibodies:

For mitochondrial membrane proteins like ATP5MG, sample preparation is particularly critical. Consider specialized extraction buffers that effectively solubilize membrane proteins while maintaining epitope integrity.

How should ATP5MG antibodies be properly stored and handled to maintain activity?

Proper storage and handling are crucial for maintaining antibody performance:

• Store at -20°C for long-term storage (most products)

• For short-term use, some products can be stored at 2-8°C for up to one month

• Aliquot antibodies upon receipt to avoid repeated freeze-thaw cycles

• Most ATP5MG antibodies are supplied in buffers containing 50% glycerol and 0.02% sodium azide as preservatives

• Always centrifuge briefly before opening vials to collect all liquid at the bottom

• Follow manufacturer's specific recommendations for each product

• Record lot numbers and maintain consistent sourcing for critical experiments

• Check expiration dates and periodically validate activity of stored antibodies

Many manufacturers provide a 12-month guarantee from the date of dispatch when antibodies are stored according to recommendations .

What controls should be included when designing experiments with ATP5MG antibodies?

Proper controls are essential for interpreting results with ATP5MG antibodies:

• Positive control: Samples known to express ATP5MG (e.g., mitochondria-rich tissues like heart, liver, or kidney)

• Negative control: Primary antibody omission control or pre-immune serum control

• Loading control: Antibody against another mitochondrial protein (for normalization)

• Specificity control: Competition with immunizing peptide (if available)

• Technical control: Replicate samples to assess reproducibility

• Biological control: Multiple biological replicates to account for natural variation

• For advanced work: siRNA knockdown or CRISPR knockout samples as definitive negative controls

• For multi-color IF/IHC: Single-staining controls to assess spectral overlap

Careful control selection helps distinguish specific signals from artifacts and ensures reliable, reproducible results in ATP5MG research.

How can ATP5MG antibodies contribute to understanding mitochondrial dysfunction in disease?

ATP5MG antibodies offer valuable tools for investigating mitochondrial dysfunction:

• Comparative analysis of ATP5MG levels in healthy versus diseased tissues can identify alterations in ATP synthase composition

• Immunohistochemical analysis can reveal changes in mitochondrial distribution and morphology

• Co-localization studies with other mitochondrial markers can assess organelle integrity

• Monitoring ATP5MG as a marker for mitochondrial stress responses

• Investigating post-translational modifications of ATP5MG in disease states

• Examining the effects of disease-associated mutations on protein-protein interactions

• Assessing the impact of therapeutic interventions on ATP synthase assembly and function

Research has demonstrated that defects in ATP synthase subunits can lead to severe multi-systemic disorders with features including hypotonia, developmental delay, and progressive encephalopathy, highlighting the clinical relevance of this research .

What experimental design considerations are important when investigating ATP5MG in complex multi-protein assemblies?

When studying ATP5MG as part of the ATP synthase complex:

• Consider using mild detergents that preserve protein-protein interactions during extraction

• Employ blue native PAGE to maintain complex integrity during electrophoretic separation

• Use crosslinking approaches to capture transient interactions

• Combine immunoprecipitation with mass spectrometry to identify interaction partners

• Consider proximity labeling techniques (BioID, APEX) with ATP5MG as bait

• Apply super-resolution microscopy to visualize spatial organization of complexes

• Use CRISPR/Cas9-mediated tagging to track ATP5MG in living cells

• Employ cryo-electron microscopy to determine structural integration of ATP5MG

These approaches can provide insights into both the static structure and dynamic assembly process of the ATP synthase complex, enhancing our understanding of mitochondrial bioenergetics in health and disease.