ATP5F1 Antibody

What is ATP5F1 and what are its key characteristics?

ATP5F1 (also known as ATP synthase subunit b) belongs to the eukaryotic ATPase B chain family. It encodes subunit B of the mitochondrial ATP synthase F0 unit, which contains a 214-amino acid protein with a 42-amino acid import signal . ATP5F1 is part of the complex F0 domain and the peripheral stalk, which acts as a stator to hold the catalytic alpha(3)beta(3) subcomplex and subunit a/ATP6 static relative to the rotary elements .

Key characteristics:

• Full Name: ATP synthase, H+ transporting, mitochondrial F0 complex, subunit B1

• Calculated Molecular Weight: 256 aa, 29 kDa

• Observed Molecular Weight: 25-30 kDa

• Gene ID (NCBI): 515

• GenBank Accession Number: BC005366

• UniProt ID: P24539

What applications can ATP5F1 antibodies be used for?

ATP5F1 antibodies have been validated for multiple applications in research:

Many commercially available antibodies have been validated for multiple applications, with Western blot being the most commonly tested application with over 21 published usages documented .

What species reactivity do ATP5F1 antibodies typically show?

ATP5F1 antibodies show reactivity with various species depending on the specific antibody:

The high sequence conservation of ATP5F1 across mammalian species often allows cross-reactivity among various species even when not specifically tested .

What are the optimal conditions for using ATP5F1 antibodies in immunohistochemistry?

For optimal IHC results with ATP5F1 antibodies:

• Dilution range: 1:100-1:400 is typically recommended

• Antigen retrieval: Use TE buffer pH 9.0; alternatively, citrate buffer pH 6.0 may be used

• Positive tissue controls: Human liver tissue, human brain tissue, human heart tissue, human kidney tissue, human skin tissue, and human testis tissue have all shown positive IHC detection

• Incubation conditions: 1 hour at room temperature is standard for primary antibody

• Detection systems: Compatible with most standard detection systems including HRP/DAB

• Counterstaining: Standard hematoxylin counterstaining is compatible

For paraffin-embedded tissues, complete deparaffinization and rehydration are essential before antigen retrieval .

How do I optimize Western blot protocols for ATP5F1 detection?

For optimal Western blot results when detecting ATP5F1:

• Sample preparation:

  • Use RIPA buffer with protease inhibitors for cell/tissue lysis

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

• Gel electrophoresis:

  • Use 12-15% SDS-PAGE gels (ATP5F1 runs at 25-30 kDa)

  • Load 10-30 μg of total protein per lane

• Transfer conditions:

  • Semi-dry or wet transfer systems are both suitable

  • Transfer at 15V for 30 minutes (semi-dry) or 100V for 1 hour (wet)

• Blocking:

  • 5% non-fat milk in TBST for 1 hour at room temperature

• Antibody dilution:

  • Primary: 1:500-1:2000 dilution in 5% BSA or milk

  • Secondary: HRP-conjugated antibody at 1:50,000-1:100,000

• Visualization:

  • Enhanced chemiluminescence (ECL) detection

  • Expected band size: 25-30 kDa

• Positive controls:

  • Human: HeLa cells, HEK-293 cells, Jurkat cells, K-562 cells

  • Mouse: heart tissue, skeletal muscle tissue, liver tissue, NIH/3T3 cells

  • Rat: HSC-T6 cells

What sample preparation methods are optimal for immunofluorescence with ATP5F1 antibodies?

For optimal immunofluorescence results:

• Cell preparation:

  • Culture cells on glass coverslips or chamber slides

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

  • Permeabilize with 0.1-0.5% Triton X-100 for 10 minutes

• Tissue preparation:

  • For blood smears: air-dry at room temperature

  • Fix overnight in 4% formaldehyde solution at 4°C

  • Wash with PBS-T (PBS with 0.05% Tween-20)

  • Perform antigen retrieval in 1mM EDTA, 0.05% Tween-20, pH 8 for 20 minutes at 95°C

• Blocking:

  • 5-10% normal serum (matching species of secondary antibody) for 1 hour

• Antibody incubation:

  • Primary: Dilute ATP5F1 antibody 1:50-1:500 in antibody diluent

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

  • Secondary: Anti-rabbit/mouse Alexa Fluor (typically 1:500-1:1000)

  • Incubate for 1 hour at room temperature

• Counterstaining:

  • DAPI (1:2000) for 10 minutes for nuclear visualization

  • Mount in fluorescence mounting media

• Controls:

  • Mitochondrial markers like VDAC1 (1:400) can be used for co-localization

  • Include a negative control by omitting primary antibody

How can ATP5F1 antibodies be used to study mitochondrial dysfunction in disease models?

ATP5F1 antibodies can be valuable tools for studying mitochondrial dysfunction:

• Expression level analysis:

  • Western blotting to quantify ATP5F1 expression changes in disease states

  • Immunohistochemistry to examine tissue-specific alterations in ATP5F1 distribution

  • Flow cytometry to assess ATP5F1 levels in specific cell populations

• ATP synthase assembly:

  • Blue Native PAGE followed by immunoblotting to analyze intact ATP synthase complex

  • Co-immunoprecipitation to study interactions with other ATP synthase subunits

  • Ratio analysis of monomer/dimer populations as indicators of mitochondrial cristae integrity

• Functional studies:

  • Combine ATP5F1 detection with enzymatic assays of ATP synthase activity

  • Correlate ATP5F1 levels with mitochondrial membrane potential measurements

  • Assess relationship between ATP5F1 expression and cellular bioenergetics

• Disease applications:

  • Neurological disorders: ATP5F1 variants have been implicated in variable neurologic phenotypes

  • Cancer metabolism: Studies show ATP5F1 participates in transcriptional and post-transcriptional regulation in cancer cells

  • Metabolic disease: Altered ATP5F1 expression correlates with changes in oxidative phosphorylation

• Therapeutic target assessment:

  • Evaluate effects of ATP5F1 inhibitors on mitochondrial function

  • Monitor ATP5F1 expression changes in response to treatment with compounds like biguanides

  • Assess knockdown/knockout phenotypes to validate therapeutic approaches

How do I validate ATP5F1 antibody specificity in my experimental system?

Comprehensive validation of ATP5F1 antibody specificity should include:

• Knockout/knockdown controls:

  • Test antibody in ATP5F1 knockout cell lines as negative controls

  • Compare signal between wild-type and siRNA/shRNA knockdown samples

  • Rescue experiments with ATP5F1 re-expression to confirm specificity

• Peptide competition assays:

  • Pre-incubate antibody with immunizing peptide before application

  • Signal should be significantly reduced or eliminated

  • Use non-specific peptide as negative control

• Multiple application validation:

  • Confirm detection across different techniques (WB, IF, IHC)

  • Verify expected subcellular localization (mitochondrial)

  • Observed molecular weight should match predicted (25-30 kDa)

• Cross-reactivity assessment:

  • Test in multiple species if working with non-human models

  • Ensure signal is absent in tissues/cells not expressing ATP5F1

  • Check for unexpected bands in Western blots

• Co-localization studies:

  • Confirm co-localization with other mitochondrial markers

  • Double-labeling with commercially validated antibodies to other ATP synthase subunits

  • Compare results with multiple ATP5F1 antibodies raised against different epitopes

What approaches are effective for studying ATP5F1's role in ATP synthase complex assembly?

To investigate ATP5F1's role in ATP synthase complex assembly:

• Native gel electrophoresis:

  • Blue Native PAGE (BN-PAGE) followed by Western blotting using ATP5F1 antibody

  • Detect shifts in ATP synthase complex size/migration

  • Monitor monomer-to-dimer ratio changes

• Co-immunoprecipitation studies:

  • Use ATP5F1 antibody for pull-down experiments

  • Identify interacting partners by mass spectrometry

  • Confirm specific interactions with other ATP synthase subunits

• Proximity labeling approaches:

  • BioID or APEX2 fusion proteins to identify proximal proteins

  • Compare interactome in normal vs. disease states

  • Identify assembly factors that interact transiently

• Structured illumination microscopy:

  • Super-resolution imaging using fluorescently labeled ATP5F1 antibodies

  • Analyze co-localization with other ATP synthase components

  • Visualize changes in mitochondrial cristae morphology

• Pulse-chase experiments:

  • Track newly synthesized ATP5F1 incorporation into the complex

  • Determine assembly kinetics and stability

  • Identify rate-limiting steps in complex formation

• CRISPR-Cas9 mutagenesis:

  • Create domain-specific mutations in ATP5F1

  • Assess effects on complex assembly and function

  • Correlate structural changes with functional outcomes

How do I troubleshoot weak or nonspecific signals when using ATP5F1 antibodies?

When encountering issues with ATP5F1 antibody signals:

For weak signals:

• Increase antibody concentration (within recommended range)

• Extend incubation time (overnight at 4°C for primary antibody)

• Optimize antigen retrieval methods (try both TE buffer pH 9.0 and citrate buffer pH 6.0)

• Use signal amplification systems (e.g., HRP-polymer detection)

• Ensure sample freshness and proper storage conditions

• Increase protein loading amount (for Western blots)

For nonspecific signals:

• Increase blocking time and concentration (5-10% normal serum)

• Use more stringent washing conditions (increase wash time and frequency)

• Reduce primary antibody concentration

• Try alternative blocking agents (5% BSA instead of milk)

• Pre-adsorb antibody with non-specific proteins

• Filter antibody solution before use (0.22 μm filter)

Application-specific troubleshooting:

• Western blot: Try different membrane types (PVDF vs. nitrocellulose)

• IHC/ICC: Optimize fixation conditions and times

• Flow cytometry: Adjust permeabilization conditions for intracellular staining

What are the critical factors to consider when comparing data from different ATP5F1 antibodies?

When comparing results from different ATP5F1 antibodies:

• Epitope differences:

  • Antibodies targeting different regions may give varying results

  • N-terminal vs C-terminal epitopes may reflect different protein populations

  • Check epitope locations: some target AA 1-245, others target middle regions (AA 161-195)

• Clonality considerations:

  • Monoclonal antibodies (like 68304-1-Ig) provide higher specificity but might miss isoforms

  • Polyclonal antibodies (like 15999-1-AP) may detect multiple isoforms/modifications

• Host species implications:

  • Most ATP5F1 antibodies are rabbit-derived, but some are mouse-derived

  • Consider secondary antibody compatibility in multi-label experiments

• Validation methods:

  • Examine how each antibody was validated (Western blot, knockout controls, etc.)

  • Look for antibodies validated in your specific application and cell/tissue type

• Buffer composition effects:

  • Different antibodies have various storage buffers (e.g., with/without glycerol, azide)

  • Buffer components may affect antibody performance in specific applications

• Post-translational modifications:

  • Some antibodies may be sensitive to specific post-translational modifications

  • Results may differ if your experimental conditions alter ATP5F1 modifications

How should I interpret changes in ATP5F1 expression in relation to mitochondrial function?

Interpreting ATP5F1 expression changes requires careful consideration:

How are ATP5F1 antibodies being used to investigate novel inhibitors and therapeutic targets?

ATP5F1 antibodies are instrumental in researching potential therapeutic approaches:

• Inhibitor mechanism studies:

  • ATP5F1 inhibitors represent a distinctive class of compounds specifically targeting ATP synthase

  • Antibodies can confirm binding and track conformational changes induced by inhibitors

  • Western blotting and immunoprecipitation can verify target engagement in treated samples

• Structure-function relationships:

  • ATP5F1 antibodies help elucidate how inhibitors disrupt proton translocation

  • Immunofluorescence tracking of protein localization following inhibitor treatment

  • Co-immunoprecipitation studies to identify altered protein interactions

• Therapeutic target validation:

  • ATP5F1 was identified as a key target of medicinal biguanides (e.g., metformin)

  • Antibodies help track ATP5F1 expression changes in response to treatment

  • In knockout systems, reintroducing ATP5F1 restores sensitivity to compounds like metformin

• Specific inhibitor examples:

  • Oligomycin A: Binds to the OSCP subunit of ATP synthase, affecting the ATP5F1 complex

  • Bongkrekic acid: Affects mitochondrial ADP/ATP exchange, indirectly impacting ATP5F1 function

  • Biotin-biguanide conjugates: Used for pull-down assays to identify ATP5F1 as a target

• Cancer metabolism applications:

  • ATP5F1 knockout in pancreatic cancer cells mimics biguanide treatment effects

  • Antibodies help characterize altered mitochondrial structure and function

  • Detecting reduced OXPHOS and increased glycolysis following ATP5F1 targeting

What are the latest techniques for analyzing ATP5F1's role in mitochondrial cristae morphology?

Advanced techniques for studying ATP5F1's role in cristae morphology include:

• Super-resolution microscopy:

  • STORM or PALM imaging with ATP5F1 antibodies to visualize distribution at nanoscale resolution

  • Live-cell STED microscopy to track dynamic changes in ATP5F1 organization

  • Correlative light and electron microscopy (CLEM) to link protein distribution with ultrastructure

• Blue Native PAGE with dimer/monomer analysis:

  • BN-PAGE followed by Western blotting using ATP5F1 antibodies

  • Quantify dimer-to-monomer ratio changes in response to treatments

  • Long-term treatments (3-6 days) with compounds like metformin can reveal subtle shifts

• Cryo-electron tomography:

  • Combine with immunogold labeling using ATP5F1 antibodies

  • 3D reconstruction of ATP synthase dimers at molecular resolution

  • Correlate structural changes with functional alterations

• Proximity labeling approaches:

  • ATP5F1 fusion with APEX2 or BioID to map protein neighborhood

  • Identify proteins involved in cristae shaping that interact with ATP5F1

  • Compare interactome in normal vs. pathological conditions

• ATP5I knockout models:

  • ATP5I (another ATP synthase subunit) stabilizes F1Fo-ATP synthase dimers

  • Compare cristae structure between ATP5F1 and ATP5I manipulations

  • Use ATP5F1 antibodies to track compensatory changes in remaining complex subunits

How can ATP5F1 antibodies be integrated with metabolomic analyses to study bioenergetic pathways?

Integrating ATP5F1 antibody techniques with metabolomics offers powerful insights:

• Multi-omics experimental design:

  • Parallel analysis of ATP5F1 protein levels and metabolite profiles

  • Correlate ATP5F1 expression (by Western blot) with ATP/ADP ratios (by LC-MS)

  • Link mitochondrial morphology (by immunofluorescence) with TCA cycle intermediates

• Isotope tracing with immunoprecipitation:

  • Combine 13C-glucose or 13C-glutamine tracing with ATP5F1 immunoprecipitation

  • Analyze protein complexes under different metabolic states

  • Correlate ATP5F1 interactome changes with metabolic flux alterations

• Spatial metabolomics integration:

  • Use ATP5F1 antibodies for tissue section immunostaining

  • Perform MALDI-MSI on adjacent sections for metabolite mapping

  • Correlate spatial distribution of ATP5F1 with energy-related metabolites

• Flux analysis with protein dynamics:

  • Measure oxygen consumption rate and extracellular acidification while tracking ATP5F1

  • Combine Seahorse analysis with immunofluorescence imaging

  • Correlate metabolic shifts with ATP5F1 expression/localization changes

• Therapeutic response monitoring:

  • Use ATP5F1 antibodies to track protein changes following metabolic interventions

  • Correlate with NAD+/NADH ratio alterations (as observed in ATP5I studies)

  • Monitor glycolytic compensation following OXPHOS inhibition