ATP5G2 Antibody

What is ATP5G2 and what cellular functions does it perform?

ATP5G2 (ATP synthase membrane subunit c locus 2, also known as ATP5MC2) is a subunit of the mitochondrial ATP synthase (Complex V). It functions as part of the F0 complex embedded in the inner mitochondrial membrane. This protein plays a critical role in ATP production by facilitating proton translocation across the membrane during oxidative phosphorylation. ATP5G2 belongs to the ATPase C chain family and forms part of the c-ring structure (likely a homomeric ring of 10 subunits) that constitutes the rotary element of the ATP synthase complex . The protein is widely expressed across human tissues and has a canonical length of 141 amino acids with a molecular mass of 14.6 kDa, though it is often observed at 28 kDa in experimental conditions .

What types of ATP5G2 antibodies are available for research and what epitopes do they target?

Several types of ATP5G2 antibodies are available for research purposes:

Researchers should select antibodies based on their experimental needs, considering whether specificity to ATP5G2 alone is required or if detection of multiple ATP5G isoforms is acceptable .

How can I validate the specificity of an ATP5G2 antibody for my experimental system?

Methodological approach to antibody validation:

• Blocking experiments: Use recombinant ATP5G2 protein fragments (such as aa 39-117) as competitive inhibitors. Pre-incubate the antibody with a 100x molar excess of the protein fragment for 30 minutes at room temperature before application in IHC/ICC or WB experiments .

• Negative controls: Include samples known to lack ATP5G2 expression or use non-immune IgG from the same species as the primary antibody.

• Knockdown/knockout validation: Compare antibody reactivity in wild-type cells versus cells where ATP5G2 has been knocked down using siRNA or knocked out using CRISPR-Cas9.

• Molecular weight verification: Confirm that the observed band corresponds to the expected molecular weight (canonical is 14.6 kDa, though ATP5G2 is often observed at 28 kDa in some experimental systems) .

• Cross-reactivity assessment: Test against related proteins, particularly ATP5G1 and ATP5G3, which share sequence homology with ATP5G2 .

What are the optimal conditions for using ATP5G2 antibodies in Western blotting?

For optimal Western blot detection of ATP5G2:

• Sample preparation:

  • Use mitochondrial enrichment protocols to increase target concentration

  • Prepare samples in reducing conditions with SDS-PAGE loading buffer

• Protein loading and separation:

  • Load 20-30 μg of total protein lysate per lane

  • Use 12-15% polyacrylamide gels for better resolution of this low molecular weight protein

• Transfer conditions:

  • Transfer to 0.2 μm nitrocellulose membranes (recommended over PVDF for this protein)

  • Use wet transfer at 100V for 1 hour or 30V overnight at 4°C

• Blocking and antibody incubation:

  • Block with 5% milk powder in TBS-T

  • For primary antibody, use dilutions of 1:1000 (for antibodies like ATP5A1) to 1:5000 (for combined ATP5G1/G2/G3 antibodies)

  • Incubate primary antibody overnight at 4°C

  • Use appropriate HRP-conjugated secondary antibodies at 1:2000-1:5000 dilution

• Detection:

  • Use enhanced chemiluminescence (ECL) detection systems

  • Exposure times typically range from 30 seconds to 5 minutes

Note that ATP5G2's observed molecular weight on Western blots (approximately 28 kDa) often differs from its calculated molecular weight (21 kDa), which is important to consider when interpreting results .

How should I optimize immunohistochemistry protocols for ATP5G2 detection in tissue sections?

Optimized IHC protocol for ATP5G2 detection:

• Tissue preparation:

  • Use 5 μm thick paraffin sections

  • Deparaffinize using xylene and rehydrate in graded ethanol

• Antigen retrieval (critical step):

  • For ATP5G2-specific antibodies, use Tris/EDTA buffer (pH 9.0)

  • Heat for 10 minutes at boiling temperature (microwave 600W)

  • Allow 30 minutes resting time followed by 15 minutes for cooling

• Blocking endogenous activity:

  • Block endogenous peroxidase with 3% hydrogen peroxide for 10 minutes

  • Wash with Tris-buffered saline containing Tween 20

• Antibody application:

  • Dilute primary antibody with Antibody diluent (e.g., Dako No. S3022)

  • Use anti-ATP5G2 at dilutions of 1:50-1:100

  • For combined anti-ATP5G1/G2/G3, use dilution of 1:100

  • Incubate at 4°C overnight

• Detection system:

  • Use HRP-labeled polymer detection systems (e.g., Dako Envision+ System)

  • Apply secondary antibodies against rabbit or mouse depending on primary

  • Counterstain with hematoxylin, dehydrate, and mount

Evaluate staining on a scale of 0-3 (0 for no staining, 3 for maximum staining) and record expression in different sub-compartments of tissues (e.g., proximal/distal tubules, collecting duct, loop of Henle in kidney tissue) .

What are the key considerations for successful immunoprecipitation experiments with ATP5G2 antibodies?

For successful IP of ATP5G2:

• Antibody selection:

  • Use antibodies specifically validated for IP (e.g., 19785-1-AP)

  • Verify reactivity with your species of interest (human, mouse)

• Sample preparation:

  • For optimal results, use HEK-293 cells (demonstrated positive IP detection)

  • Use gentle lysis buffers that preserve protein-protein interactions

  • Include protease inhibitors to prevent degradation

• Antibody amount:

  • Use 0.5-4.0 μg of antibody for every 1.0-3.0 mg of total protein lysate

  • Pre-clear lysate with protein A/G beads to reduce non-specific binding

• Incubation conditions:

  • Incubate antibody with lysate overnight at 4°C with gentle rotation

  • Use protein A or protein G sepharose beads depending on antibody host species

  • Wash beads thoroughly (4-5 times) with wash buffer

• Elution and analysis:

  • Elute proteins using either SDS-sample buffer at 95°C or low pH

  • Analyze by Western blot using another ATP5G2 antibody (recognizing a different epitope)

  • Include appropriate negative controls (non-specific IgG, lysate without antibody)

For co-immunoprecipitation studies investigating ATP5G2's interaction partners within the ATP synthase complex, consider using cross-linking reagents to stabilize transient interactions.

How can I differentiate between ATP5G2 and other ATP synthase subunits (particularly ATP5G1 and ATP5G3) in my experiments?

Differentiation strategies:

• Antibody selection:

  • Use ATP5G2-specific antibodies rather than those recognizing multiple subunits

  • Confirm specificity using immunoblotting against recombinant ATP5G1, ATP5G2, and ATP5G3 proteins

• PCR-based approaches:

  • Design primers specific to unique regions of ATP5G2 mRNA

  • Validate primer specificity using plasmids containing each isoform

• Expression pattern analysis:

  • ATP5G1 shows stronger down-regulation (fold change -8.09) in ccRCC compared to ATP5G2 (fold change -2.69) and ATP5G3 (fold change -3.10)

  • Use this differential expression pattern in certain tissues to identify isoforms

• Mass spectrometry:

  • Identify isoform-specific peptides for unambiguous identification

  • Use targeted MS approaches for higher sensitivity

• Genetic manipulation:

  • Use isoform-specific siRNA knockdown followed by antibody detection

  • Create isoform-specific knockout cell lines using CRISPR-Cas9

When using combined antibodies (detecting ATP5G1/G2/G3), complementary molecular approaches are necessary to distinguish between isoforms .

What factors might affect ATP5G2 antibody performance and how can I mitigate these issues?

Common factors affecting antibody performance and mitigation strategies:

Regular validation of antibody performance using positive and negative controls is essential for reliable results.

What are the critical differences in methodology when using ATP5G2 antibodies across different species samples?

Methodological considerations for cross-species applications:

• Sequence homology assessment:

  • Human ATP5G2 shows 57% antigen sequence identity with mouse and rat orthologs

  • This moderate homology suggests potential cross-reactivity but necessitates validation

• Antibody selection:

  • Choose antibodies validated for your species of interest

  • Some antibodies (e.g., 19785-1-AP) are validated for both human and mouse samples

• Protocol adjustments:

  • Antigen retrieval: May need species-specific optimization

  • Antibody concentration: Often requires higher concentrations for less homologous species

  • Incubation time: Consider extending for cross-species applications

• Control samples:

  • Include species-specific positive controls

  • Consider using tissues/cells with known high ATP5G2 expression for each species

• Alternative approaches for poorly conserved regions:

  • Use antibodies targeting highly conserved epitopes

  • Consider combined antibodies against ATP5G1/G2/G3 when studying evolutionary aspects

• Validation methods:

  • Perform peptide competition assays with species-specific ATP5G2 peptides

  • Compare with species-specific mRNA expression data

When working with non-human samples, preliminary validation experiments are crucial to confirm antibody reactivity and specificity.

How can ATP5G2 antibodies be used to investigate mitochondrial dysfunction in disease models?

Advanced methodological approaches:

• Subcellular localization studies:

  • Use ATP5G2 antibodies in combination with other mitochondrial markers

  • Perform co-localization analyses using confocal microscopy

  • Quantify changes in localization pattern during disease progression

• Complex V assembly analysis:

  • Use ATP5G2 antibodies in blue native PAGE experiments

  • Investigate alterations in ATP synthase assembly in disease conditions

  • Combine with antibodies against other subunits to assess stoichiometry changes

• Post-translational modifications:

  • Use phospho-specific or other modification-specific antibodies

  • Combine with mass spectrometry to identify novel modifications in disease states

• Mitochondrial dynamics assessment:

  • Correlate ATP5G2 expression with mitochondrial morphology and distribution

  • Study the relationship between ATP5G2 levels and mitochondrial fission/fusion proteins

• In vivo disease models:

  • Use ATP5G2 antibodies to track mitochondrial changes in:

    • Neurodegenerative disease models (particularly Batten disease)

    • Cancer models (especially ccRCC which shows ATP5G2 downregulation)

    • Metabolic disease models

• Therapeutic response monitoring:

  • Assess changes in ATP5G2 expression/localization following treatment

  • Use as a biomarker for mitochondrial recovery in intervention studies

This approach is particularly relevant for Batten disease research, as ATP5G2 is the major protein stored in the storage bodies of affected individuals .

What experimental approaches can elucidate the role of ATP5G2 in clear cell renal cell carcinoma (ccRCC) progression?

• Tissue microarray analysis:

  • Use ATP5G2 antibodies on ccRCC tumor microarrays with patient outcome data

  • Score staining intensity (0-3 scale) and correlate with clinical parameters

  • Compare with normal renal tissue compartments (proximal/distal tubules, collecting duct)

• Expression correlation studies:

  • Combine ATP5G2 IHC with markers of hypoxia, angiogenesis, and mTOR pathway

  • Analyze correlations between ATP5G2 levels and these pathways

  • Create multivariate models for prediction of patient outcomes

• Functional studies in ccRCC cell lines:

  • Modulate ATP5G2 expression (overexpression/knockdown)

  • Assess effects on:

    • Cell proliferation and apoptosis

    • Migration and invasion capacity

    • Metabolic reprogramming (Warburg effect)

    • Response to targeted therapies

• ATP synthase activity assays:

  • Correlate ATP5G2 protein levels with functional ATP synthase activity

  • Compare enzymatic activity between normal kidney and ccRCC tissues

  • Assess relationship between activity and tumor aggressiveness

• Multi-omics integration:

  • Correlate ATP5G2 protein levels with transcriptomics and metabolomics data

  • Identify molecular signatures associated with ATP5G2 expression

How can quantitative proteomics be combined with ATP5G2 antibody-based approaches to study mitochondrial ATP synthase complex stoichiometry?

Integrated methodological workflow:

• Sample preparation:

  • Isolate intact mitochondria from tissues/cells of interest

  • Perform gentle solubilization to maintain complex integrity

  • Fractionate samples to enrich ATP synthase complex components

• Antibody-based complex isolation:

  • Use ATP5G2 antibodies for immunoprecipitation of intact ATP synthase complex

  • Alternatively, use antibodies against tagged ATP5G2 in systems with tagged protein expression

  • Include appropriate controls (non-specific IgG, lysate without antibody)

• Mass spectrometry analysis:

  • Perform tryptic digestion of immunoprecipitated complex

  • Use label-free or isotope labeling approaches for quantification

  • Identify unique peptides for each subunit for accurate quantification

• Data analysis and interpretation:

  • Calculate relative abundances of complex components

  • Compare stoichiometric ratios across different conditions/tissues

  • Identify condition-specific alterations in complex composition

• Validation experiments:

  • Confirm key findings using orthogonal approaches

  • Use Western blotting with antibodies against multiple subunits

  • Perform blue native PAGE to assess complex integrity

• Integration with functional data:

  • Correlate stoichiometric changes with ATP synthase activity measurements

  • Assess impact of altered stoichiometry on mitochondrial function

This integrated approach provides deeper insights into ATP synthase complex dynamics than either antibody-based or proteomics methods alone.

What is the relationship between ATP5G2 expression and Batten disease, and how can antibodies help study this connection?

ATP5G2 has significant relevance to Batten disease (ceroid lipofuscinosis) research:

• Pathophysiological significance:

  • ATP5G2 is the major protein stored in the storage bodies of animals or humans affected with ceroid lipofuscinosis

  • This accumulation suggests a fundamental role in disease pathogenesis

• Research methodologies using ATP5G2 antibodies:

  • Tissue analysis: Use antibodies to quantify ATP5G2 accumulation in different brain regions and correlate with symptom severity

  • Subcellular localization: Determine whether ATP5G2 mislocalizes to lysosomes or other organelles in disease state

  • Animal models: Track disease progression by monitoring ATP5G2 aggregation using immunohistochemistry

  • Post-translational modifications: Investigate whether disease-specific modifications of ATP5G2 contribute to aggregation

• Therapeutic development applications:

  • Use ATP5G2 antibodies to screen compounds that prevent protein aggregation

  • Monitor treatment efficacy by quantifying changes in ATP5G2 accumulation

  • Develop assays to measure clearance of ATP5G2 aggregates following intervention

• Diagnostic potential:

  • Evaluate ATP5G2 antibodies for detecting early disease biomarkers

  • Develop quantitative assays to measure ATP5G2 levels in accessible biofluids

• Mechanistic studies:

  • Investigate whether ATP5G2 aggregation leads to mitochondrial dysfunction

  • Examine interactions between ATP5G2 and lysosomal proteins in disease conditions

This research direction could significantly advance understanding of Batten disease pathogenesis and identify new therapeutic targets.

How can ATP5G2 antibodies be used to investigate the relationship between mitochondrial dysfunction and cancer?

Comprehensive research approach:

This approach leverages the established link between ATP5G2 expression and patient outcomes in ccRCC and expands investigation to other cancer types.

What methodologies can resolve the apparent discrepancy between ATP5G2's calculated molecular weight (21 kDa) and its observed weight in experimental conditions (28 kDa)?

Systematic investigative approach:

• Protein sequence analysis:

  • Examine the full sequence of ATP5G2 including potential alternative splicing variants

  • Analyze the presence of signal peptides or pro-protein sequences

  • Calculate theoretical weights of different isoforms (3 known isoforms exist)

• Post-translational modification investigation:

  • Use phospho-specific antibodies to detect potential phosphorylation

  • Employ glycosylation-specific staining methods

  • Perform enzymatic deglycosylation experiments and observe mobility shifts

  • Mass spectrometry analysis to identify and map modifications

• Experimental verification:

  • Treat samples with various denaturing conditions to assess protein folding effects

  • Compare migration patterns under reducing vs. non-reducing conditions

  • Express recombinant ATP5G2 with and without tags to assess mobility

• Comparative analysis:

  • Compare migration patterns of ATP5G2 across different tissues and species

  • Assess whether the discrepancy is consistent across experimental systems

  • Compare with ATP5G1 and ATP5G3 migration patterns

• Sample preparation effects:

  • Evaluate different protein extraction methods

  • Assess the impact of buffer composition on observed molecular weight

  • Test effects of different detergents on membrane protein solubilization

• Analytical approach verification:

  • Use alternative molecular weight determination methods (e.g., gel filtration)

  • Compare results from different gel percentage and buffer systems

  • Employ mass spectrometry for absolute molecular weight determination

This comprehensive approach will help determine whether the observed discrepancy represents biological reality (due to modifications or structural features) or is an artifact of experimental conditions.

How can ATP5G2 antibodies be used in conjunction with super-resolution microscopy to study mitochondrial ultrastructure?

Advanced imaging methodological approach:

• Sample preparation for super-resolution microscopy:

  • Optimize fixation protocols to preserve mitochondrial ultrastructure

  • Use ATP5G2 antibodies conjugated to fluorophores suitable for STORM/PALM/STED

  • Perform dual labeling with outer membrane markers for contextual information

• Imaging protocol development:

  • Use STORM/PALM for single-molecule localization (~20 nm resolution)

  • Apply STED microscopy for live-cell imaging of ATP5G2 dynamics

  • Implement Expansion Microscopy for physical magnification of structures

• Data acquisition and analysis:

  • Collect 3D image stacks to reconstruct mitochondrial architecture

  • Perform drift correction and multi-color channel alignment

  • Use computational approaches to extract quantitative spatial information

• Biological applications:

  • Map ATP5G2 distribution within cristae membranes at nanoscale resolution

  • Investigate reorganization of ATP synthase complexes during mitochondrial stress

  • Examine ATP5G2 clustering in disease models (particularly Batten disease)

  • Assess ATP5G2 distribution changes in ccRCC compared to normal kidney tissue

• Correlative approaches:

  • Combine super-resolution fluorescence with electron microscopy

  • Correlate ATP5G2 nanoscale distribution with functional parameters

  • Integrate with live-cell imaging to link structure and dynamics

This cutting-edge approach would reveal previously unobservable details about ATP5G2's role in mitochondrial structure and function, with particular relevance to disease states where ATP5G2 expression is altered.

What is the potential for developing ATP5G2-targeted therapeutic approaches, and how can antibodies facilitate this research?

Research framework for therapeutic development:

• Target validation studies:

  • Use ATP5G2 antibodies to confirm expression in disease-relevant tissues

  • Perform knockdown/overexpression studies to establish phenotypic consequences

  • Correlate expression with disease progression in patient samples

• Functional screening platforms:

  • Develop cell-based assays measuring ATP5G2 expression/localization

  • Create reporter systems for ATP synthase activity

  • Establish high-content screening approaches using ATP5G2 antibodies

• Therapeutic modalities exploration:

  • Small molecules: Screen compounds that stabilize ATP synthase assembly

  • Biologics: Develop therapeutic antibodies targeting accessible epitopes

  • Gene therapy: Use antibodies to monitor expression from gene therapy vectors

  • RNA therapeutics: Design antisense oligonucleotides or siRNAs and monitor effects

• Disease-specific applications:

  • Batten disease: Test approaches to prevent ATP5G2 accumulation in storage bodies

  • ccRCC: Explore methods to restore ATP5G2 expression, given its correlation with survival

  • Other mitochondrial disorders: Target ATP5G2 to stabilize ATP synthase function

• Companion diagnostic development:

  • Use ATP5G2 antibodies to develop assays predicting treatment response

  • Create standardized IHC protocols for patient stratification

Therapeutic targeting of ATP5G2 represents a novel approach that could address fundamental aspects of mitochondrial dysfunction in multiple diseases.

How can single-cell proteomics approaches incorporating ATP5G2 antibodies advance our understanding of mitochondrial heterogeneity?

Methodological workflow for single-cell ATP5G2 analysis:

• Sample preparation optimization:

  • Develop protocols for single-cell isolation preserving mitochondrial integrity

  • Optimize fixation and permeabilization for antibody accessibility

  • Establish multiplexing approaches for simultaneous detection of multiple targets

• Single-cell proteomics techniques:

  • Mass cytometry (CyTOF): Use metal-conjugated ATP5G2 antibodies

  • Single-cell Western blotting: Capture individual cells and perform protein separation

  • Microfluidic antibody capture: Isolate single cells in droplets with barcoded antibodies

  • In situ PLA (Proximity Ligation Assay): Detect ATP5G2 interactions at single-molecule resolution

• Data acquisition and analysis:

  • Apply dimensionality reduction techniques (tSNE, UMAP) to visualize cell populations

  • Perform clustering analyses to identify cell subsets with distinct ATP5G2 expression

  • Integrate with single-cell transcriptomics data for multi-omic insights

• Biological applications:

  • Map ATP5G2 expression heterogeneity across individual cells in tissues

  • Identify rare cell populations with abnormal ATP5G2 expression

  • Track changes in ATP5G2 expression during cellular differentiation or disease progression

• Tissue-specific investigations:

  • Analyze renal cell populations for ATP5G2 heterogeneity relevant to ccRCC

  • Examine neuronal populations for ATP5G2 variations related to Batten disease

  • Study cancer cell populations to identify metabolically distinct subsets

This cutting-edge approach would reveal previously unappreciated cellular heterogeneity in ATP5G2 expression and function, with implications for understanding disease mechanisms and developing targeted therapies.