ATP5C1 Antibody

What is ATP5C1 and why is it significant for research?

ATP5C1 (ATP synthase subunit gamma, mitochondrial) is also known as ATP5C and ATP5CL1, belonging to the ATPase gamma chain family. It functions as a critical component of the mitochondrial F1 complex responsible for ATP synthesis. With an observed molecular weight of 33 kDa, this protein plays an essential role in cellular energy production through oxidative phosphorylation .

Research significance includes:

• Central role in mitochondrial bioenergetics

• Involvement in metabolic disorders and neurodegenerative diseases

• Potential biomarker for mitochondrial dysfunction

• Dysregulation linked to various pathological conditions including cancer

The protein's location in the mitochondrial inner membrane makes it valuable for studying mitochondrial structure and function, while its high conservation across species facilitates comparative studies between human and animal models .

What types of ATP5C1 antibodies are available for research?

Researchers have multiple options when selecting ATP5C1 antibodies:

Polyclonal antibodies like 10910-1-AP offer broader epitope recognition and strong signal amplification, making them suitable for detection of native proteins across multiple species . The monoclonal antibody 60284-1-Ig provides higher specificity but with more limited species reactivity, primarily human samples . Both antibody types are generated using recombinant fusion proteins containing ATP5C1 sequences as immunogens .

What are the validated applications for ATP5C1 antibodies?

ATP5C1 antibodies have been validated across multiple experimental applications:

For immunohistochemistry applications, antigen retrieval with TE buffer pH 9.0 is recommended, though citrate buffer pH 6.0 may serve as an alternative . The diversity of validated applications makes these antibodies versatile tools for comprehensive protein analysis across multiple experimental contexts.

What are the optimal dilutions for different applications?

Proper antibody dilution is critical for balancing signal strength and background. Recommended dilutions vary by application:

It is recommended to titrate these antibodies in each testing system to obtain optimal results, as optimal dilution can be sample-dependent . Starting with the middle of the recommended range and adjusting based on signal-to-noise ratio is a prudent approach for optimization.

What positive controls should be used with ATP5C1 antibodies?

Selecting appropriate positive controls ensures experimental validity:

For 10910-1-AP (polyclonal):

• Western Blot: Mouse brain tissue, human heart tissue, human/mouse skeletal muscle tissue, rat brain/skeletal muscle tissue

• IHC: Human liver cancer tissue

• IF/ICC: HepG2 cells

For 60284-1-Ig (monoclonal):

• Western Blot: Human heart tissue, fetal human brain tissue

• IHC: Human liver cancer tissue

• IF/ICC: HepG2 cells

• Flow Cytometry: HeLa cells

For CAB15257 (polyclonal):

• Western Blot: Mouse heart, mouse brain

These validated positive controls have consistently demonstrated reliable ATP5C1 detection and should be incorporated into experimental design to verify antibody performance.

How should ATP5C1 antibodies be stored and handled?

Proper storage and handling are essential for maintaining antibody performance:

Storage conditions:

• Store at -20°C in provided buffer (PBS with 0.02% sodium azide and 50% glycerol, pH 7.3)

• Stable for one year after shipment when properly stored

• Aliquoting is unnecessary for -20°C storage

• Some preparations (20μL sizes) contain 0.1% BSA

Handling recommendations:

• Avoid repeated freeze-thaw cycles

• Allow antibody to equilibrate to room temperature before opening

• Centrifuge briefly before use to collect solution at the bottom of the tube

• When diluting, use appropriate buffers compatible with the intended application

• For long-term storage of diluted antibody, add preservatives like sodium azide to prevent microbial growth

Following these guidelines ensures maximum antibody performance and extended shelf-life.

How can ATP5C1 antibodies be used to study mitochondrial dysfunction?

ATP5C1 antibodies serve as powerful tools for investigating mitochondrial dysfunction in various disease models:

Experimental approaches:

• Protein expression analysis: Western blotting to quantify ATP5C1 levels in control vs. disease samples, revealing alterations in mitochondrial ATP synthase components

• Subcellular localization studies: Immunofluorescence to examine potential mislocalization of ATP5C1 in diseased cells

• Tissue distribution profiling: IHC to map ATP5C1 expression patterns across healthy and pathological tissues

• Protein-protein interaction studies: Co-immunoprecipitation with ATP5C1 antibodies to identify altered binding partners in dysfunction states

ATP5C1 dysregulation has been linked to various diseases, including metabolic disorders, neurodegenerative conditions, and cancer . By measuring changes in ATP5C1 expression, researchers can gain insights into mitochondrial bioenergetic alterations underlying these pathologies.

What are best practices for optimizing Western blots with ATP5C1 antibodies?

Achieving optimal Western blot results with ATP5C1 antibodies requires attention to several critical factors:

Sample preparation:

• Use appropriate lysis buffers containing protease inhibitors

• Enrich for mitochondrial fractions when studying low-abundance samples

• Denature samples at 95°C for 5 minutes in reducing sample buffer

Electrophoresis and transfer:

• Use 10-12% SDS-PAGE gels for optimal resolution of the 33 kDa ATP5C1 protein

• Transfer to nitrocellulose or PVDF membranes at 100V for 60-90 minutes

• Verify transfer efficiency with reversible protein stains

Immunodetection:

• Block with 5% non-fat milk or 3-5% BSA in TBST for 1 hour at room temperature

• Incubate with primary antibody (1:500-1:3000 for 10910-1-AP; 1:500-1:2000 for 60284-1-Ig) overnight at 4°C

• Wash thoroughly with TBST (3-5 times, 5-10 minutes each)

• Incubate with appropriate HRP-conjugated secondary antibody

• Develop using enhanced chemiluminescence detection

For enhanced sensitivity, as demonstrated with the 2I2 ScFv antibody in teucrin A studies, optimized antibody dilutions can detect as little as 2.5 ng of adducted protein with brief exposure times .

What troubleshooting approaches address common issues with ATP5C1 antibodies?

Researchers may encounter several challenges when working with ATP5C1 antibodies:

For antigen retrieval in IHC applications, note that both TE buffer (pH 9.0) and citrate buffer (pH 6.0) have been validated, with the former being the primary recommendation . When analyzing complex samples, researchers should be aware that high molecular weight immunoreactive bands may result from protein contaminants or aggregates, as observed in Western blot analyses of BSA samples .

How do monoclonal and polyclonal ATP5C1 antibodies compare in research applications?

Understanding the comparative advantages of monoclonal and polyclonal ATP5C1 antibodies helps researchers select the optimal tool:

Polyclonal ATP5C1 antibodies (10910-1-AP, CAB15257):

• Recognize multiple epitopes on ATP5C1, enhancing detection sensitivity

• Show broader species cross-reactivity (human, mouse, rat)

• Particularly effective for detecting native proteins in applications like IP

• Ideal for proteins with low expression levels

• May show batch-to-batch variation

Monoclonal ATP5C1 antibody (60284-1-Ig):

• Recognizes a single epitope, providing higher specificity

• More limited species reactivity (primarily human)

• Produces consistent results across experiments

• Lower background in certain applications

• Well-suited for flow cytometry applications

Selection considerations should include the specific experimental question, required species reactivity, and application type. For studies requiring detection of subtle changes in ATP5C1 levels across multiple species, polyclonal antibodies may be preferable. For highly specific detection in human samples with minimal background, the monoclonal option offers advantages.

How are ATP5C1 antibodies used in immunohistochemistry of tissue samples?

Effective IHC protocols for ATP5C1 detection in tissues include these key steps:

• Sample preparation:

  • Fix tissues in 10% neutral buffered formalin

  • Embed in paraffin and section at 4-6 μm thickness

  • Mount sections on positively charged slides

• Antigen retrieval:

  • Primary recommendation: TE buffer pH 9.0

  • Alternative: Citrate buffer pH 6.0

  • Heat-induced epitope retrieval methods (pressure cooker, microwave, or water bath)

• Immunostaining protocol:

  • Block endogenous peroxidase activity with 3% H₂O₂

  • Block non-specific binding with serum-free protein block

  • Apply ATP5C1 antibody at appropriate dilution (1:250-1:1000 for 10910-1-AP; 1:100-1:400 for 60284-1-Ig)

  • Incubate overnight at 4°C or 1-2 hours at room temperature

  • Apply appropriate detection system (e.g., HRP-polymer)

  • Develop with DAB and counterstain with hematoxylin

• Controls and validation:

  • Positive control: Human liver cancer tissue

  • Negative controls: Primary antibody omission and isotype controls

  • Expected pattern: Mitochondrial/cytoplasmic staining

ATP5C1 antibodies have been successfully used to study mitochondrial distribution and abundance in various tissues, providing insights into tissue-specific energy requirements and pathological alterations.

What are the optimal protocols for immunofluorescence studies with ATP5C1 antibodies?

For cellular localization studies using immunofluorescence:

• Cell preparation:

  • Culture cells on coverslips or chamber slides

  • Fix with 4% paraformaldehyde for 15 minutes at room temperature

  • Permeabilize with 0.2% Triton X-100 for 5-10 minutes

• Immunostaining protocol:

  • Block with 1-5% BSA or normal serum in PBS for 30-60 minutes

  • Incubate with ATP5C1 antibody (1:200-1:800 for 10910-1-AP; 1:50-1:500 for 60284-1-Ig)

  • Incubate overnight at 4°C or 1-2 hours at room temperature

  • Wash with PBS (3 times, 5 minutes each)

  • Apply fluorophore-conjugated secondary antibody

  • Counterstain nuclei with DAPI or similar nuclear stain

  • Mount with anti-fade mounting medium

• Validated cell lines:

  • HepG2 cells have been validated for both polyclonal and monoclonal ATP5C1 antibodies

  • Expected pattern shows punctate cytoplasmic staining consistent with mitochondrial localization

For co-localization studies, ATP5C1 antibodies can be paired with other mitochondrial markers such as TOMM20 (outer membrane) or COX4 (inner membrane) to investigate mitochondrial structural integrity and protein distribution within the organelle.

How can ATP5C1 antibodies be incorporated into flow cytometry protocols?

Flow cytometry with ATP5C1 antibodies enables quantitative analysis of protein expression across cell populations:

• Sample preparation:

  • Harvest cells using appropriate methods (trypsinization, scraping)

  • Wash cells in PBS

  • Fix with 2-4% paraformaldehyde for 10-15 minutes

  • Permeabilize with 0.1-0.5% saponin or 0.1% Triton X-100

• Staining protocol:

  • Block with 5% normal serum in permeabilization buffer

  • Apply ATP5C1 antibody (0.40 μg per 10^6 cells for 60284-1-Ig)

  • Incubate for 30-60 minutes at room temperature

  • Wash twice with permeabilization buffer

  • Apply fluorophore-conjugated secondary antibody

  • Incubate for 30 minutes at room temperature

  • Wash and resuspend in appropriate buffer for analysis

• Controls and validation:

  • Isotype control: Mouse IgG2a for 60284-1-Ig

  • Positive control: HeLa cells

  • Gating strategy: Exclude debris and doublets, then analyze ATP5C1 expression

This approach enables quantitative assessment of ATP5C1 expression across different cell types or experimental conditions, particularly useful for studying mitochondrial alterations in heterogeneous cell populations or disease models.

How are ATP5C1 antibodies used to investigate mitochondrial disease mechanisms?

ATP5C1 antibodies provide valuable insights into mitochondrial disease pathology through several research approaches:

• Expression profiling across disease states:

  • Western blot analysis to quantify ATP5C1 levels in patient-derived samples versus controls

  • IHC to map ATP5C1 distribution in affected tissues

  • Flow cytometry to measure expression in specific cell populations

• Functional correlation studies:

  • Pairing ATP5C1 expression data with mitochondrial functional assays (oxygen consumption, ATP production)

  • Correlating ATP5C1 levels with disease severity or progression markers

• Therapeutic response monitoring:

  • Tracking ATP5C1 expression changes in response to mitochondrial-targeted therapies

  • Using ATP5C1 as a biomarker for treatment efficacy

ATP5C1 dysregulation has been linked to various pathological conditions including metabolic disorders, neurodegenerative diseases, and cancer . By measuring the expression and localization of this key mitochondrial protein, researchers can better understand disease mechanisms related to bioenergetic dysfunction.

What considerations are important when using ATP5C1 antibodies in protein interaction studies?

Investigating ATP5C1 protein interactions requires careful experimental design:

• Immunoprecipitation approaches:

  • Use antibody 10910-1-AP for IP applications (validated in publications)

  • Employ gentle lysis conditions to preserve native protein complexes

  • Pre-clear lysates to reduce non-specific binding

  • Include appropriate negative controls (isotype-matched IgG)

• Cross-linking considerations:

  • Consider reversible cross-linkers to stabilize transient interactions

  • Optimize cross-linking conditions to balance complex preservation with epitope accessibility

• Mass spectrometry integration:

  • After immunoprecipitation with ATP5C1 antibodies, samples can be analyzed by LC-MS/MS

  • For complex samples, be aware that high molecular weight bands may represent protein aggregates or contaminants

• Validation strategies:

  • Confirm interactions with reciprocal IP experiments

  • Verify co-localization using immunofluorescence microscopy

  • Consider functional assays to establish biological relevance of identified interactions

These approaches enable researchers to map the ATP5C1 interactome and identify alterations in protein-protein interactions that may contribute to mitochondrial dysfunction in various disease contexts.