atp5if1a Antibody
Description
Definition and Target Protein Overview
ATP5IF1 (ATP Synthase Inhibitory Factor 1), also known as IF1, is a 12.2 kDa protein encoded by the ATP5IF1 gene in humans. It consists of 106 amino acids and localizes to mitochondria, where it reversibly inhibits ATP synthase activity during hypoxia or low pH, preventing ATP hydrolysis and preserving cellular energy .
Biological Functions
ATP5IF1 regulates metabolic reprogramming in activated T cells and adaptive immunity:
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Metabolic Regulation: Inhibits ATP synthase to shift cells toward glycolysis, essential for T-cell activation .
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Immune Function: Required for Th1 effector cell differentiation; knockout models show impaired immune responses to bacterial infections .
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Mitochondrial Biogenesis: Promotes mitochondrial replication during T-cell proliferation .
Research Applications of ATP5IF1 Antibodies
Anti-ATP5IF1 antibodies are widely used in:
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Western Blot: Detects endogenous ATP5IF1 in mitochondrial lysates .
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Immunohistochemistry (IHC): Localizes ATP5IF1 in tissue sections .
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Immunoprecipitation (IP): Isolates ATP5IF1-protein complexes .
Role in T-Cell Immunity
CD4+-IF1-KO mice (T-cell-specific ATP5IF1 knockout) exhibit:
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Impaired Glycolysis: Reduced glucose uptake and lactate production .
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Defective Proliferation: 50% lower T-cell expansion in vitro .
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Susceptibility to Infection: 100% mortality in Listeria-infected KO mice vs. 80% survival in wild-type .
Mechanistic Insights
| Observation | Wild-Type vs. KO | Citation |
|---|---|---|
| Glycolytic flux | 2.5-fold higher in WT | |
| Mitochondrial biogenesis | 40% reduction in KO | |
| IFNγ production (Th1 cells) | 70% decrease in KO |
Therapeutic Implications
ATP5IF1 is a potential target for modulating immune responses:
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Autoimmunity: Suppressing IF1 could reduce pathogenic Th1 activity.
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Cancer Immunotherapy: Enhancing IF1 might boost T-cell metabolic fitness against tumors .
Unresolved Questions
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How does ATP5IF1 expression vary across T-cell subsets (e.g., Th17 vs. Treg)?
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Can small-molecule inhibitors of ATP5IF1 enhance anti-tumor immunity?
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- The identification of mitochondrial Atpif1 as a regulator of heme synthesis provides valuable insights into the mechanisms governing mitochondrial heme homeostasis and red blood cell development. PMID: 23135403
Q&A
What is ATP5IF1 and what is its biological function?
ATP5IF1 (ATP synthase inhibitory factor subunit 1) is a small protein that serves as a natural inhibitor of mitochondrial ATP synthase. In humans, the canonical form consists of 106 amino acid residues with a molecular weight of approximately 12.2 kDa . It is primarily localized in the mitochondria and belongs to the ATPase inhibitor protein family. ATP5IF1 plays a critical role in energy metabolism regulation by inhibiting the hydrolytic activity of ATP synthase during conditions of low oxygen, thereby preventing ATP depletion.
Beyond its primary role in bioenergetic regulation, ATP5IF1 has been implicated in angiogenesis and erythrocyte differentiation processes . This protein is widely expressed across multiple tissue types and demonstrates high evolutionary conservation, with orthologs identified in numerous species including mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken .
What are the common applications for ATP5IF1 antibodies in research?
ATP5IF1 antibodies serve diverse research applications, each providing unique insights into protein expression, localization, and function:
When selecting an application, researchers should consider the specific research question, available sample types, and the validated applications for their particular antibody.
How do you validate the specificity of an ATP5IF1 antibody?
Thorough validation of ATP5IF1 antibody specificity is essential for generating reliable research data. A comprehensive validation approach should include:
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Positive control testing: Use samples known to express ATP5IF1, such as heart mitochondria from humans, bovine, mouse, or rat .
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Western blot analysis: Confirm detection of a protein at the expected molecular weight (approximately 12.2 kDa for canonical ATP5IF1) .
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Knockout/knockdown validation: Compare antibody reactivity between wild-type samples and those with reduced ATP5IF1 expression through genetic modification or RNA interference.
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Peptide competition assay: Pre-incubate the antibody with excess ATP5IF1 peptide (immunogen) to block specific binding sites; this should substantially reduce signal if the antibody is specific.
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Cross-reactivity assessment: Test the antibody against related proteins to ensure it doesn't detect other members of the ATPase inhibitor protein family.
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Multiple antibody approach: Use different antibodies targeting distinct epitopes of ATP5IF1 to confirm consistent results.
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Method-specific controls: For each application, include appropriate technical controls (e.g., primary antibody omission, isotype controls).
Detailed documentation of validation procedures and results is essential for publication quality and experimental reproducibility.
What species reactivity can be expected with ATP5IF1 antibodies?
ATP5IF1 demonstrates high evolutionary conservation, enabling many antibodies to recognize the protein across multiple species. Based on available commercial antibodies, the following species reactivity has been documented:
When selecting an ATP5IF1 antibody, researchers should verify the specific species reactivity claimed by manufacturers and consider conducting their own validation if working with less common species. Some antibodies may cross-react with predicted species including pig, chicken, and Xenopus based on sequence homology .
What are the differences between monoclonal and polyclonal ATP5IF1 antibodies?
The choice between monoclonal and polyclonal ATP5IF1 antibodies significantly impacts experimental outcomes. Understanding their distinct characteristics helps researchers select the optimal reagent for specific applications:
Application-specific considerations:
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For detecting denatured ATP5IF1 in Western blots, both types work well, but monoclonals often provide cleaner results
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For detecting native conformations, epitope accessibility becomes critical
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For detecting specific isoforms, monoclonals targeting unique regions are preferred
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For applications requiring signal amplification, polyclonals may be advantageous
The selection should be guided by the specific research application, available budget, and whether batch-to-batch consistency is critical for longitudinal studies.
How do ATP5IF1 expression levels vary across different tissue types?
ATP5IF1 exhibits tissue-specific expression patterns that reflect differential energy requirements and metabolic activities across organs:
When studying ATP5IF1 expression, researchers should employ appropriate technical approaches:
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Quantitative Western blotting: Using ATP5IF1 antibodies with proper loading controls such as GAPDH
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Immunohistochemistry: For spatial distribution within tissues
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qRT-PCR: To correlate protein levels with mRNA expression
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ELISA: For quantitative comparison across sample types
These tissue-specific differences in ATP5IF1 expression likely reflect adaptation to varying energy demands and may indicate tissue-specific regulatory mechanisms controlling mitochondrial ATP synthesis.
What are the known isoforms of ATP5IF1 and how can antibodies differentiate between them?
Up to three different isoforms of ATP5IF1 have been reported in humans . Distinguishing between these isoforms requires careful antibody selection and methodological approaches:
| Approach | Methodology | Advantages | Limitations |
|---|---|---|---|
| Epitope-specific antibodies | Antibodies targeting unique sequences in specific isoforms | Direct discrimination between isoforms | Requires known sequence differences |
| High-resolution Western blot | SDS-PAGE with extended run times | Can separate subtle molecular weight differences | May not resolve post-translational modifications |
| 2D electrophoresis | Separation by both isoelectric point and molecular weight | Can differentiate modifications and variants | Technically challenging |
| Immunoprecipitation + Mass spectrometry | Pull-down followed by peptide analysis | Definitive identification of isoforms | Requires specialized equipment |
| Recombinant isoform controls | Expression of each isoform for antibody validation | Provides clear specificity profile | Requires molecular cloning capabilities |
When studying ATP5IF1 isoforms, researchers should:
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Determine which isoform(s) their antibody detects based on epitope information
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Consider how isoform differences might affect experimental interpretation
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Use complementary approaches to confirm isoform-specific findings
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Document which isoform(s) are being studied in publications
This attention to isoform specificity is particularly important when comparing results across different studies or tissue types where isoform expression may vary.
What experimental controls should be included when using ATP5IF1 antibodies?
Robust experimental design incorporating appropriate controls is essential for generating reliable and interpretable results with ATP5IF1 antibodies:
For quantitative applications, standard curves using recombinant ATP5IF1 protein should be included. For comparative studies, all samples must be processed identically to minimize technical variability.
Additionally, when studying mitochondrial proteins like ATP5IF1, appropriate fractionation controls should verify the purity of mitochondrial preparations and account for potential changes in mitochondrial content between samples.
How can ATP5IF1 antibodies be used to investigate mitochondrial dysfunction in neurodegenerative diseases?
Mitochondrial dysfunction is a hallmark of many neurodegenerative diseases, and ATP5IF1 antibodies provide valuable tools for investigating these pathological processes:
| Research Approach | Methodology | Insights Provided |
|---|---|---|
| Expression profiling | Western blotting and IHC in disease models and patient samples | Reveals altered ATP5IF1 expression patterns in affected brain regions |
| Bioenergetic correlation | Combine antibody detection with functional assays (oxygen consumption, ATP production) | Links ATP5IF1 expression to energy deficits |
| Protein aggregation studies | Co-immunofluorescence with disease-specific aggregates (tau, amyloid, α-synuclein) | Examines potential interactions with pathological proteins |
| Oxidative stress response | Track ATP5IF1 expression changes after oxidative challenge | Elucidates protective mechanisms |
| Mitochondrial morphology | Super-resolution microscopy with ATP5IF1 antibodies | Visualizes structural alterations in diseased mitochondria |
| Mitophagy assessment | Co-staining with autophagy markers | Evaluates mitochondrial quality control mechanisms |
Experimental approaches should include:
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Comparison between affected and unaffected brain regions
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Age-matched controls to account for age-related changes
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Correlation with disease severity metrics
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Longitudinal studies in animal models to track disease progression
ATP5IF1's role extends beyond ATP synthase regulation, as it participates in transcriptional and post-transcriptional regulation of genes associated with glucose homeostasis and HIF-1 signaling , which are often dysregulated in neurodegenerative conditions.
How can ATP5IF1 antibodies be used to investigate the role of this protein in cancer progression?
ATP5IF1 has emerging roles in cancer biology that can be investigated using antibody-based approaches:
Research findings indicate that ATP5IF1 overexpression significantly increases expression of genes associated with the innate immune response, angiogenesis, and collagen catabolic processes, including matrix metalloproteinases MMP2 and MMP19 . Additionally, ATP5IF1 can interfere with alternative splicing of hundreds of genes linked to glucose homeostasis, HIF-1 signaling activation, and several cancer-associated pathways .
This multifaceted approach using various antibody applications can provide comprehensive insights into how ATP5IF1 contributes to cancer initiation, progression, and therapeutic response across different cancer types.
How does ATP5IF1 regulate mitochondrial ATP synthase activity and how can this be studied with antibodies?
ATP5IF1 is a natural inhibitor of mitochondrial ATP synthase, playing a crucial role in cellular energy homeostasis. Antibody-based approaches offer valuable tools to elucidate this regulatory mechanism:
| Regulatory Aspect | Antibody-Based Methodology | Mechanistic Insights |
|---|---|---|
| Physical interaction | Co-immunoprecipitation with ATP5IF1 antibodies | Identifies conditions promoting/disrupting ATP5IF1-ATP synthase binding |
| Inhibitory mechanism | In vitro activity assays after antibody-mediated depletion | Quantifies direct effects on ATP synthase function |
| Stress response | Proximity ligation assays during hypoxia/pH changes | Reveals dynamic interactions under stress conditions |
| Conformational changes | Conformation-specific antibodies | Distinguishes active vs. inactive ATP5IF1 forms |
| Oligomerization | Native gel electrophoresis with Western blotting | Examines formation of regulatory ATP5IF1 dimers/oligomers |
| Subcellular localization | Immunofluorescence and sub-mitochondrial fractionation | Maps precise location within mitochondrial compartments |
When designing experiments to study ATP5IF1's regulatory role:
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Include appropriate physiological stressors (hypoxia, pH changes) that modulate ATP5IF1 activity
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Consider the dynamic, reversible nature of the inhibition
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Account for potential post-translational modifications that regulate binding
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Correlate binding with functional outcomes (ATP synthesis/hydrolysis rates)
This comprehensive approach provides insights into how ATP5IF1 helps maintain cellular energy homeostasis, particularly during stress conditions when preventing ATP depletion becomes critical for cell survival.
What techniques can be used to analyze ATP5IF1 post-translational modifications using antibodies?
Post-translational modifications (PTMs) of ATP5IF1 can significantly alter its function, localization, and regulatory capacity. Several antibody-based techniques can be employed to study these modifications:
| Technique | Methodology | Advantages | Limitations |
|---|---|---|---|
| Modification-specific antibodies | Western blot/IHC/IF with phospho-, acetyl- or ubiquitin-specific antibodies | Direct detection of specific modifications | Requires available modification-specific antibodies |
| 2D-Gel Electrophoresis | Separation by isoelectric point and molecular weight followed by Western blotting | Can separate modified forms | Labor-intensive, requires optimization |
| Immunoprecipitation + PTM detection | Pull-down ATP5IF1 followed by PTM-specific antibody probing | Enriches target protein before analysis | May lose transient modifications |
| IP + Mass Spectrometry | Antibody purification followed by MS analysis | Comprehensive PTM mapping | Requires specialized equipment |
| Proximity Ligation Assay | Combine ATP5IF1 and PTM-specific antibodies | In situ visualization of modifications | Semi-quantitative |
| Flow Cytometry | Dual staining with ATP5IF1 and PTM antibodies | Quantifies modified populations | Limited spatial information |
| ELISA-based PTM quantification | Capture with ATP5IF1 antibody, detect with PTM antibody | High-throughput quantification | May have sensitivity limitations |
When studying ATP5IF1 PTMs, researchers should:
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Consider physiological conditions that might trigger modifications (hypoxia, pH changes)
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Include appropriate controls (phosphatase/deacetylase treatments)
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Confirm findings using complementary techniques
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Correlate modifications with functional outcomes
This multi-technique approach provides comprehensive insights into how PTMs regulate ATP5IF1 function in different cellular contexts and disease states.
What are the challenges in developing antibodies against highly conserved proteins like ATP5IF1?
Developing high-quality antibodies against evolutionary conserved proteins like ATP5IF1 presents several significant challenges:
| Challenge | Technical Implication | Potential Solutions |
|---|---|---|
| Limited immunogenicity | Weak immune response in host animals | Use synthetic peptides or fusion proteins as immunogens |
| Epitope selection difficulties | Finding unique regions for specificity | Detailed sequence analysis across species; target less conserved regions |
| Cross-reactivity concerns | Non-specific binding to related proteins | Extensive validation against related family members |
| Species-reactivity tradeoffs | Balancing specificity vs. broad reactivity | Consider research needs when selecting epitope regions |
| Tolerance mechanisms | Host immune system may not respond to conserved antigens | Use carrier proteins; alternative host species |
| Isoform discrimination | Distinguishing between ATP5IF1 isoforms | Target isoform-specific sequences when possible |
| Validation complexity | Demonstrating true specificity | Comprehensive validation across applications and species |
Generating antibodies against highly conserved antigens like ATP5IF1 requires specialized approaches to overcome these challenges . Techniques such as phage display technology can help circumvent immunological tolerance issues . When developing antibodies against such "difficult antigens," researchers must employ rigorous validation strategies to ensure both specificity and sensitivity in the intended applications.
How can ATP5IF1 antibodies be used to study mitochondrial function?
ATP5IF1 antibodies provide valuable tools for investigating various aspects of mitochondrial biology and function:
| Research Area | Antibody-Based Methodology | Mitochondrial Function Insights |
|---|---|---|
| ATP synthase regulation | Co-IP to isolate ATP5IF1-ATP synthase complexes | Elucidates inhibitory mechanism under different conditions |
| Bioenergetic profiling | Western blotting correlated with functional assays | Links expression to ATP production capacity |
| Mitochondrial morphology | IF microscopy with mitochondrial network markers | Reveals relationship between ATP5IF1 and structural organization |
| Hypoxia response | Track expression changes during oxygen limitation | Examines adaptive mechanisms to metabolic stress |
| Tissue energy metabolism | IHC across tissue types | Correlates with tissue-specific energy requirements |
| Mitochondrial disorders | Expression analysis in disease models | Identifies potential biomarkers or therapeutic targets |
| Mitophagy and quality control | Dual IF with autophagy markers | Reveals role in mitochondrial turnover |
When designing such studies, researchers should:
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Include appropriate mitochondrial loading controls (e.g., ATP5A1 or VDAC)
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Consider the impact of mitochondrial content variations between samples
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Correlate protein-level findings with functional outcomes
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Account for potential compensatory mechanisms
ATP5A1, another subunit of the ATP synthase complex, is frequently used as a control in ATP5IF1 studies and can be detected using specific antibodies that recognize this approximately 60 kDa protein . This complementary approach provides context for understanding ATP5IF1's role within the larger ATP synthase complex.
