Atp5f1b Antibody

What is ATP5F1B and why is it important in research?

ATP5F1B (previously known as ATP5B) is the beta subunit of mitochondrial ATP synthase (Complex V) of the respiratory chain. This 56.56 kDa protein forms part of the catalytic core in F1, with subunits alpha and beta creating the site for ATP synthesis. During catalysis, ATP synthesis in the F1 domain couples with proton translocation through a rotary mechanism .

ATP5F1B is crucial for understanding mitochondrial function, energy metabolism, and has been implicated in several diseases including neurological disorders. Recent research has identified variants in ATP5F1B associated with dominantly inherited dystonia .

What applications are ATP5F1B antibodies validated for?

ATP5F1B antibodies have been validated for multiple applications across different experimental contexts:

The optimal dilution should be determined experimentally as it may vary depending on sample type and detection method .

What species reactivity can be expected from ATP5F1B antibodies?

Most commercially available ATP5F1B antibodies demonstrate cross-reactivity with multiple species due to the high conservation of this protein:

When selecting an antibody for your research, verify the validated species reactivity and consider sequence homology if working with non-validated species .

What are optimal sample preparation protocols for ATP5F1B detection?

Sample preparation is critical for successful ATP5F1B detection:

For Western Blotting:

• Harvest cells or tissues and lyse in a buffer containing protease inhibitors

• For mitochondrial protein enrichment, perform subcellular fractionation

• Denature samples at 95°C for 5 minutes in SDS loading buffer

• Load 10-30 μg of protein per lane on SDS-PAGE (10-12%)

• Transfer to PVDF membranes (preferred over nitrocellulose for mitochondrial proteins)

• Block with 5% non-fat milk or BSA in TBST

Observed molecular weight is typically 52-56 kDa depending on the experimental system .

For Immunocytochemistry:

• Fix cells with 4% paraformaldehyde (10-15 minutes)

• For optimal mitochondrial staining, permeabilize with 0.1% Triton X-100

• Block with 1-5% BSA or serum

• Incubate with primary antibody overnight at 4°C

• Counter-stain with mitochondrial markers (e.g., MitoTracker) for co-localization studies

• Use DAPI for nuclear counterstaining

How should ATP5F1B antibodies be stored and handled to maintain activity?

Proper storage and handling are essential for maintaining antibody activity:

• Store at -20°C for long-term storage (up to one year)

• For frequent use, store at 4°C for up to one month

• Avoid repeated freeze-thaw cycles as this degrades antibody quality

• Aliquot antibodies upon receipt to minimize freeze-thaw cycles

• Most formulations contain 50% glycerol, PBS, and a preservative (sodium azide or proclin-300)

• Reconstitute lyophilized antibodies in sterile distilled water with 50% glycerol

Note that sodium azide inhibits HRP activity, so ensure thorough washing when using HRP-conjugated secondary antibodies with primary antibodies containing sodium azide .

How can ATP5F1B antibodies be used to investigate mitochondrial dysfunction in neurodegenerative disorders?

ATP5F1B antibodies are valuable tools for investigating mitochondrial dysfunction in neurodegenerative disorders:

• Assessment of Complex V assembly and integrity:

  • Using blue native PAGE (BN-PAGE) with ATP5F1B antibodies can reveal abnormal high molecular weight bands corresponding to dimeric or oligomeric complex V, or to complex V-containing super-complexes, as observed in patients with ATP5F1B variants

• Determination of mitochondrial membrane potential:

  • ATP5F1B antibodies can be used alongside JC-1 staining to correlate Complex V dysfunction with membrane potential changes

  • In mutant fibroblasts with ATP5F1B variants, areas of green JC-1 monomeric staining indicate reduced membrane potential

• Investigation of dystonia mechanisms:

  • Recent research has identified ATP5F1B variants (p.Thr334Pro and p.Val482Ala) in families with early-onset isolated dystonia

  • ATP5F1B antibodies can be used to determine if protein levels are altered in patient samples

  • Functional studies using these antibodies have revealed that dystonia-associated mutations cause severe reduction of complex V activity despite normal protein levels, suggesting a dominant-negative effect

What methodological approaches can be used to investigate ATP5F1B in the context of mitochondrial super-complexes?

Investigating ATP5F1B in super-complexes requires specialized techniques:

• Blue Native PAGE (BN-PAGE):

  • Solubilize mitochondria with mild detergents (digitonin preferred over Triton X-100)

  • Run samples on gradient gels (3-12% or 4-16%) at 4°C

  • Transfer to PVDF membranes using tank transfer

  • Probe with ATP5F1B antibodies to detect monomeric, dimeric, and super-complex forms

  • In pathological conditions, abnormal high molecular weight bands may appear

• Co-immunoprecipitation with crosslinking:

  • Use membrane-permeable crosslinkers to stabilize transient interactions

  • Immunoprecipitate with ATP5F1B antibodies

  • Analyze interacting partners by mass spectrometry or Western blotting

  • This can identify novel interactions between Complex V and other respiratory chain complexes

• Proximity labeling approaches:

  • Express ATP5F1B fused to promiscuous biotin ligases (BioID or TurboID)

  • Identify proximal proteins by streptavidin pulldown and mass spectrometry

  • Validate interactions using ATP5F1B antibodies

These approaches have revealed that mutations in ATP5F1B can affect super-complex formation, potentially impacting energy production efficiency .

What are common sources of non-specific binding when using ATP5F1B antibodies and how can they be mitigated?

Non-specific binding with ATP5F1B antibodies can arise from several sources:

• Cross-reactivity with other F1F0 ATPase subunits:

  • ATP5F1B shares structural similarities with other ATP synthase subunits

  • Solution: Validate antibody specificity using knockout/knockdown controls

  • Perform peptide competition assays to confirm binding specificity

• High background in immunostaining:

  • Often due to inadequate blocking or excessive antibody concentration

  • Solution: Optimize blocking (try 2-5% BSA or serum)

  • Increase washing time and volume

  • Titrate antibody concentration to determine optimal dilution

• Multiple bands in Western blot:

  • May represent degradation products or post-translational modifications

  • Solution: Use fresh samples with protease inhibitors

  • Include phosphatase inhibitors if studying phosphorylated forms

  • Validate bands using recombinant ATP5F1B or samples from knockout models

• Non-specific nuclear or cytoplasmic staining:

  • May occur if mitochondrial membranes are compromised

  • Solution: Optimize fixation conditions (try 4% PFA for 10-15 minutes)

  • Use methanol fixation (100% methanol, 10 minutes at -20°C) for better mitochondrial preservation

How can ATP5F1B antibody specificity be verified in experimental settings?

Verifying antibody specificity is crucial for reliable results:

• Positive and negative control samples:

  • Use cell lines with confirmed high expression (HeLa, HepG2, 293T cells show strong ATP5F1B expression)

  • Compare with tissues known to have different expression levels (high in heart, liver, brain; lower in non-metabolically active tissues)

  • If possible, include ATP5F1B-knockdown or knockout samples

• Peptide competition assays:

  • Pre-incubate the antibody with the immunizing peptide

  • A specific antibody will show reduced or eliminated signal

• Western blot validation:

  • The expected molecular weight for ATP5F1B is approximately 56 kDa, though 52 kDa is commonly observed on SDS-PAGE

  • Multiple antibodies targeting different epitopes should detect the same protein band

• Immunofluorescence co-localization:

  • ATP5F1B should co-localize with mitochondrial markers

  • Compare staining patterns with established mitochondrial markers (MitoTracker, TOMM20, etc.)

  • Counterstain nuclei with DAPI to confirm mitochondrial localization

How do ATP5F1B variants contribute to dystonia pathogenesis?

Recent research has identified ATP5F1B variants associated with dystonia that provide insights into disease mechanisms:

• Dominant inheritance pattern:

  • Two different ATP5F1B missense variants (c.1000A>C; p.Thr334Pro and c.1445T>C; p.Val482Ala) have been found in families with early-onset isolated dystonia

  • These variants show autosomal dominant inheritance with incomplete penetrance

• Functional consequences:

  • Fibroblasts from patients with ATP5F1B mutations show:

    • Normal or increased ATP5F1B protein levels (no haploinsufficiency)

    • Severe and isolated ATPase deficiency

    • Impaired mitochondrial membrane potential

    • Formation of abnormal high-molecular-weight complex V structures

  • These findings suggest a dominant-negative mechanism rather than loss of function

• Tissue specificity:

  • Despite ATP5F1B being ubiquitously expressed, mutations selectively affect the nervous system

  • This may be due to the high energy demands of neurons and their sensitivity to bioenergetic deficits

  • Research using ATP5F1B antibodies can help explain this selective vulnerability by comparing ATP5F1B complex formation in different tissues

What methodological approaches can be used to investigate the relationship between ATP5F1B dysfunction and mitochondrial membrane potential?

Investigating this relationship requires specialized techniques:

• JC-1 staining with ATP5F1B co-localization:

  • JC-1 shows red fluorescence in mitochondria with high membrane potential and green in depolarized mitochondria

  • Co-staining with ATP5F1B antibodies can reveal correlations between ATP5F1B distribution and membrane potential

  • In fibroblasts with ATP5F1B mutations, areas with green JC-1 staining indicate reduced membrane potential

• Live-cell imaging with membrane potential sensors:

  • Use TMRM or other potential-sensitive dyes alongside transfected fluorescent-tagged ATP5F1B

  • This allows real-time monitoring of potential changes in relation to ATP5F1B distribution

• Patch-clamp techniques with immunocytochemistry:

  • Measure membrane potential directly with patch-clamp

  • Follow with ATP5F1B immunostaining to correlate electrophysiological data with protein localization

• Pharmacological manipulation:

  • Apply complex V inhibitors (oligomycin) or uncouplers (FCCP)

  • Compare effects in wild-type versus cells expressing mutant ATP5F1B

  • Monitor changes using ATP5F1B antibodies in fixed cells or real-time with tagged constructs

These approaches have revealed that ATP5F1B mutations can impair membrane potential without affecting oxygen consumption, suggesting alternative mechanisms for energy production may be activated .

How can ATP5F1B antibodies contribute to therapeutic development for mitochondrial disorders?

ATP5F1B antibodies serve crucial roles in therapeutic development:

• Target validation:

  • Confirm expression and localization of ATP5F1B in disease models

  • Determine if therapeutic candidates restore normal ATP5F1B distribution and function

  • Monitor changes in complex V assembly and activity in response to treatments

• Biomarker development:

  • Assess whether circulating ATP5F1B levels correlate with disease state

  • Determine if post-translational modifications of ATP5F1B could serve as diagnostic markers

  • Develop assays to detect ATP5F1B in extracellular vesicles as potential biomarkers

• High-throughput screening:

  • Develop cell-based assays using ATP5F1B antibodies to screen compound libraries

  • Identify molecules that normalize complex V assembly or activity

  • Screen for compounds that prevent dominant-negative effects of mutant ATP5F1B

• Personalized medicine approaches:

  • Use ATP5F1B antibodies to characterize patient-derived cells

  • Determine if specific mutations affect ATP5F1B localization, complex assembly, or activity

  • Develop mutation-specific therapeutic strategies based on molecular phenotyping

Research using ATP5F1B antibodies has already revealed that dystonia-associated mutations cause a dominant-negative effect rather than haploinsufficiency, suggesting potential therapeutic approaches aimed at counteracting this mechanism rather than simply increasing protein expression .

What emerging techniques could enhance ATP5F1B antibody-based research?

Several cutting-edge approaches show promise for advancing ATP5F1B research:

• Super-resolution microscopy:

  • Techniques like STED, PALM, and STORM can resolve ATP5F1B distribution within mitochondrial cristae

  • Visualize interactions between ATP5F1B and other complex V subunits at nanometer resolution

  • Correlate structural changes with functional deficits in disease models

• Cryo-electron tomography:

  • Combining ATP5F1B immunogold labeling with cryo-ET can reveal complex V organization in situ

  • Provides structural insights into how mutations affect ATP synthase assembly and organization

• Single-cell proteomics:

  • Analyze ATP5F1B expression and post-translational modifications at single-cell resolution

  • Understand cell-to-cell variability in ATP5F1B function and its relevance to disease

• CRISPR-based approaches:

  • Generate precise ATP5F1B mutations to model disease variants

  • Create reporter systems by tagging endogenous ATP5F1B with fluorescent proteins

  • Develop CRISPR interference/activation systems to modulate ATP5F1B expression

These emerging techniques will provide unprecedented insights into ATP5F1B biology and its role in health and disease .

What are the most pressing unanswered questions regarding ATP5F1B function and dysfunction?

Several critical questions remain to be addressed:

• Structural effects of disease mutations:

  • How do ATP5F1B mutations alter the molecular structure of complex V?

  • Do these structural changes affect interactions with other respiratory chain complexes?

  • Can structural alterations be targeted therapeutically?

• Tissue-specific consequences:

  • Why do ubiquitous ATP5F1B mutations primarily affect the nervous system?

  • Are there tissue-specific interaction partners that modulate ATP5F1B function?

  • Do compensatory mechanisms exist in unaffected tissues?

• Role in aging and neurodegeneration:

  • How does ATP5F1B function change during normal aging?

  • Is ATP5F1B dysfunction a common pathway in multiple neurodegenerative diseases?

  • Could targeting ATP5F1B function slow neurodegenerative processes?

• Post-translational regulation:

  • How is ATP5F1B activity regulated by post-translational modifications?

  • Are these modifications altered in disease states?

  • Can these modifications be therapeutically targeted?