ATPAF1 Antibody, HRP conjugated

What are the optimal storage conditions for ATPAF1 Antibody, HRP conjugated?

ATPAF1 Antibody, HRP conjugated should be stored at -20°C or -80°C upon receipt. It is critical to avoid repeated freeze-thaw cycles as this can compromise antibody activity and stability . The antibody is typically supplied in a storage buffer containing preservatives (such as 0.03% Proclin 300) and stabilizers (50% Glycerol, 0.01M PBS, pH 7.4) . For long-term storage, aliquoting the antibody into smaller volumes is recommended to minimize freeze-thaw cycles. When handling the antibody, it should be kept on ice and returned to storage promptly after use. The stability is generally guaranteed for one year after shipment when stored properly.

What species reactivity can be expected with ATPAF1 antibodies?

The species reactivity of ATPAF1 antibodies varies depending on the specific product and manufacturer. Based on available data, certain ATPAF1 antibodies (such as the E-9 clone) demonstrate reactivity with ATPAF1 from multiple species including mouse, rat, and human origins . In contrast, some other products like the Cusabio HRP-conjugated polyclonal antibody (CSB-PA002424LB01HU) are specifically designed for human ATPAF1 detection only . When planning experiments involving different species, researchers should carefully verify the cross-reactivity information provided by the manufacturer and consider conducting preliminary validation tests if working with species not explicitly listed in the product specifications.

What are the validated applications for ATPAF1 Antibody, HRP conjugated?

ATPAF1 Antibody, HRP conjugated has been validated for enzyme-linked immunosorbent assay (ELISA) applications according to manufacturer specifications . Other non-HRP conjugated ATPAF1 antibodies have demonstrated utility in multiple experimental applications including:

When using the HRP-conjugated format, researchers should note that while it is primarily optimized for ELISA, it may also be suitable for direct detection in Western blotting without the need for secondary antibodies, potentially reducing background and cross-reactivity issues .

How should ATPAF1 Antibody, HRP conjugated be optimized for Western blotting experiments?

Although the HRP-conjugated ATPAF1 antibody is primarily validated for ELISA, it can be adapted for Western blotting by following these optimization steps:

• Sample preparation: For optimal detection of mitochondrial proteins like ATPAF1, use appropriate mitochondrial isolation protocols to enrich for the target protein. ATPAF1 is expressed in the mitochondrial matrix, so proper fractionation techniques will improve detection sensitivity.

• Dilution optimization: Begin with a 1:500 dilution and adjust as needed based on signal intensity and background levels. A titration experiment (testing dilutions from 1:250 to 1:2000) is recommended for first-time use with your specific samples.

• Blocking optimization: Use 5% non-fat dry milk or 3-5% BSA in TBST (Tris-buffered saline with 0.1% Tween-20) for blocking. Test both to determine which provides optimal signal-to-noise ratio.

• Incubation conditions: Incubate membrane with antibody solution at 4°C overnight with gentle agitation for best results. For rapid protocols, room temperature incubation for 1-2 hours may be sufficient but may result in higher background.

• Signal development: Since the antibody is HRP-conjugated, use enhanced chemiluminescence (ECL) substrate directly without secondary antibody. Exposure times should be optimized based on the abundance of the target protein.

• Controls: Include positive controls (cell lines known to express ATPAF1, such as HeLa cells) and negative controls (ATPAF1 knockdown samples if available) .

What is the recommended protocol for validating ATPAF1 antibody specificity?

A comprehensive validation strategy for ATPAF1 antibody specificity should include:

• Knockout/knockdown validation: Generate ATPAF1 knockout or knockdown cell lines (using CRISPR-Cas9 or siRNA) and confirm loss of signal in Western blotting, immunofluorescence, or ELISA.

• Peptide competition assay: Pre-incubate the antibody with excess immunizing peptide (if available) before application to the sample. Specific binding should be blocked by this competition.

• Molecular weight verification: Confirm that the detected band in Western blot corresponds to the expected molecular weight of ATPAF1 (approximately 12-14 kDa, depending on post-translational modifications).

• Co-localization studies: In immunofluorescence experiments, confirm co-localization with established mitochondrial markers (such as MitoTracker or antibodies against other mitochondrial proteins).

• Multiple antibody verification: Compare results using antibodies from different suppliers or those raised against different epitopes of ATPAF1.

• Mass spectrometry validation: For definitive confirmation, immunoprecipitate ATPAF1 and analyze by mass spectrometry to confirm identity.

• Cross-species reactivity testing: If the antibody is claimed to work across species, verify specificity in each species of interest separately .

How can ATPAF1 Antibody, HRP conjugated be used to investigate mitochondrial dysfunction in disease models?

ATPAF1 Antibody, HRP conjugated can be instrumental in investigating mitochondrial dysfunction in various disease models through these advanced applications:

• Quantitative ELISA for ATPAF1 expression levels: Develop a quantitative ELISA using the HRP-conjugated antibody to precisely measure ATPAF1 levels in patient samples or disease model systems. Changes in ATPAF1 expression could indicate alterations in ATP synthase assembly efficiency.

• ATP synthase assembly monitoring: Use the antibody in combination with blue native PAGE and immunoblotting to assess the assembly state of ATP synthase complexes in disease models. This approach can reveal whether assembly defects contribute to the pathology.

• Tissue-specific expression analysis: Apply immunohistochemistry techniques (after methodological adaptation of the HRP-conjugated antibody) to examine tissue-specific expression patterns of ATPAF1 in disease versus healthy tissues.

• High-throughput screening: Develop cell-based assays using the antibody for high-throughput screening of compounds that might affect ATP synthase assembly in disease models.

• Correlation studies: Correlate ATPAF1 levels with clinical parameters, ATP production capacity, and other mitochondrial function markers to establish pathophysiological relationships in metabolic diseases.

• Co-immunoprecipitation studies: Adapt the antibody for co-IP experiments to investigate potential altered protein-protein interactions involving ATPAF1 in pathological conditions .

What are the considerations when using ATPAF1 Antibody, HRP conjugated in multiplex assays with other mitochondrial markers?

When incorporating ATPAF1 Antibody, HRP conjugated into multiplex assays with other mitochondrial markers, researchers should consider:

• Detection system compatibility: The HRP conjugate generates a signal that may overlap with other peroxidase-based detection systems. Consider using fluorescent reporters for other targets or sequential detection protocols.

• Cross-reactivity assessment: Perform thorough controls to ensure no cross-reactivity between the ATPAF1 antibody and other mitochondrial proteins, especially those involved in ATP synthase assembly (e.g., ATPAF2, ATP5A1, ATP5B).

• Epitope accessibility: In complex multiplex assays, epitope masking can occur due to steric hindrance or competition between antibodies. Optimize antibody concentrations and incubation sequences.

• Signal normalization: Include appropriate housekeeping controls specific to mitochondria (such as VDAC or mitochondrial HSP60) for accurate normalization when quantifying multiple targets.

• Compartment-specific controls: Include markers for different mitochondrial compartments (matrix, inner membrane, intermembrane space, outer membrane) to properly contextualize ATPAF1 localization and abundance.

• Signal amplification balance: When combining with non-HRP conjugated antibodies, ensure that signal amplification methods are balanced to prevent over-representation of highly abundant proteins .

How does ATPAF1 interact with the ATP synthase complex during assembly, and how can the antibody help elucidate this process?

ATPAF1 functions as a dedicated assembly factor for the F1 component of ATP synthase, specifically facilitating the incorporation of F1-beta and F1-alpha subunits. The ATPAF1 Antibody, HRP conjugated can be employed in several sophisticated experimental approaches to elucidate this process:

• Sequential immunoprecipitation: Use the antibody in a modified IP protocol to capture ATPAF1-bound intermediates of ATP synthase assembly, followed by mass spectrometry analysis to identify the exact composition of assembly intermediates.

• Pulse-chase assays: Combine radioactive labeling of newly synthesized proteins with immunoprecipitation using the ATPAF1 antibody to track the temporal dynamics of ATP synthase assembly.

• Proximity ligation assays: Adapt the antibody for proximity ligation assays to visualize and quantify interactions between ATPAF1 and various ATP synthase subunits in situ.

• Protein-RNA interaction studies: Recent research suggests potential RNA involvement in mitochondrial protein import and assembly. The antibody could be used in RNA immunoprecipitation assays to identify RNAs that might interact with ATPAF1 during assembly, similar to findings with ATP5A1 .

• Structural studies: Use the antibody to purify native ATPAF1-containing complexes for cryo-electron microscopy analysis to determine the structural basis of assembly factor function.

• In vitro reconstitution: Combine the antibody with in vitro translation systems to monitor the assembly process in a controlled environment and identify rate-limiting steps .

What are common troubleshooting strategies for weak or non-specific signals when using ATPAF1 Antibody, HRP conjugated?

When encountering weak or non-specific signals with ATPAF1 Antibody, HRP conjugated, consider these troubleshooting approaches:

• Sample preparation issues:

  • Ensure complete lysis of mitochondria using appropriate detergents (e.g., digitonin or Triton X-100)

  • Verify protein integrity by checking for degradation with Coomassie staining

  • Include protease inhibitors in all buffers to prevent target degradation

• Antibody-specific issues:

  • Test different dilutions (start with manufacturer recommendations, then adjust)

  • Reduce incubation temperature (4°C overnight instead of room temperature)

  • Verify antibody activity with a positive control sample

  • Check antibody storage conditions and expiration date

• Detection system issues:

  • Use fresh ECL substrate and ensure proper mixing of components

  • Optimize exposure times for chemiluminescence detection

  • Try enhanced detection systems (e.g., super-signal substrates) for low-abundance targets

• Non-specific binding resolution:

  • Increase blocking agent concentration (5-10% milk or BSA)

  • Add 0.1-0.5% Tween-20 to antibody dilution buffer

  • Include 0.1-0.3M NaCl in washing and antibody dilution buffers to reduce ionic interactions

  • Pre-adsorb antibody with cell/tissue lysate from a source lacking the target

• Cross-reactivity management:

  • Validate with knockout/knockdown controls

  • Run parallel blots with non-immune IgG to identify non-specific bands

  • Use antigen-specific peptide blocking to identify specific signal

How should researchers interpret and quantify ATPAF1 expression data across different tissue types or experimental conditions?

When analyzing ATPAF1 expression data across different tissues or experimental conditions, consider these interpretation and quantification guidelines:

• Normalization approaches:

  • For tissue comparisons: Normalize to mitochondrial mass markers (e.g., VDAC, HSP60) rather than whole-cell housekeeping genes

  • For experimental manipulations: Use ratio to baseline rather than absolute values

  • Consider multiple normalization references and compare results for robustness

• Statistical analysis recommendations:

  • For multiple tissue comparisons: Use ANOVA with appropriate post-hoc tests

  • For paired treatments: Apply paired t-tests or Wilcoxon signed-rank tests as appropriate

  • Consider non-parametric tests when sample sizes are small or normality cannot be assumed

• Biological context considerations:

  • Interpret ATPAF1 changes in context of ATP synthase abundance

  • Correlate with functional measures (e.g., ATP production, oxygen consumption)

  • Consider tissue-specific baseline differences in mitochondrial content

• Visualization strategies:

  • Present data as fold-change with error bars

  • Consider heat maps for multi-tissue comparisons

  • Include representative images/blots alongside quantification

• Contradictory data handling:

  • When results conflict with literature, verify antibody specificity

  • Consider tissue-specific post-translational modifications affecting recognition

  • Evaluate whether methodological differences explain discrepancies

What experimental controls are essential when studying ATPAF1 in relation to ATP synthase assembly defects?

When investigating ATPAF1's role in ATP synthase assembly defects, these controls are essential:

• Biological controls:

  • Positive control: Cell line with known ATPAF1 expression (e.g., HeLa cells)

  • Negative control: ATPAF1 knockdown/knockout samples

  • Reference control: Cell line with characterized ATP synthase assembly (for comparison)

• Technical controls:

  • Loading control: Mitochondrial marker independent of ATP synthase (e.g., VDAC)

  • Antibody specificity control: Pre-immunization serum or isotype control

  • Secondary antibody control: Sample processed without primary antibody

• Functional controls:

  • ATP synthase activity measurements (e.g., oligomycin-sensitive ATPase activity)

  • Oxygen consumption rate measurements

  • Membrane potential assessments (using fluorescent dyes like TMRM)

• Assembly state controls:

  • Blue native PAGE of fully assembled ATP synthase complex

  • Immunoprecipitation of ATP synthase subunits to verify interactions

  • Analysis of other assembly factors (e.g., ATPAF2) for comparison

• Rescue experiments:

  • Complementation with wild-type ATPAF1 in deficient cells

  • Domain mutation studies to identify critical regions

  • Species-specific rescue to assess evolutionary conservation

How is ATPAF1 antibody research contributing to our understanding of mitochondrial diseases?

ATPAF1 antibody research is advancing our understanding of mitochondrial diseases through several important avenues:

• Diagnostic biomarker development: ATPAF1 antibodies are enabling the development of quantitative assays that may serve as biochemical markers for specific mitochondrial disorders characterized by ATP synthase assembly defects.

• Pathogenic mechanism elucidation: By studying ATPAF1's interactions during ATP synthase assembly, researchers are uncovering molecular mechanisms underlying mitochondrial dysfunction in diseases. The antibody allows visualization and quantification of assembly intermediates that accumulate in pathological conditions.

• Genotype-phenotype correlation studies: ATPAF1 antibodies are helping researchers correlate specific genetic variants in ATPAF1 or related genes with altered protein expression, stability, or function, providing insights into why certain mutations cause disease.

• Therapeutic target identification: By precisely characterizing ATPAF1's role in ATP synthase assembly, researchers can identify potential points of intervention for developing therapies aimed at enhancing or supplementing defective assembly processes.

• Drug screening platforms: ATPAF1 antibodies are being incorporated into high-throughput screening platforms to identify compounds that might enhance ATP synthase assembly in disease models.

• Tissue-specific manifestations: The antibody is helping reveal tissue-specific differences in ATPAF1 expression and function, potentially explaining why certain tissues are more affected in mitochondrial diseases .

What are the latest techniques for studying protein-protein interactions involving ATPAF1 and ATP synthase components?

Cutting-edge techniques for investigating protein-protein interactions between ATPAF1 and ATP synthase components include:

• Proximity-dependent biotin identification (BioID): By fusing ATPAF1 to a biotin ligase, researchers can identify proteins that come into close proximity during the assembly process through biotinylation and subsequent purification.

• CRISPR-based screening: CRISPR interference or activation screens, coupled with ATPAF1 antibody-based readouts, can identify genetic modifiers of ATP synthase assembly.

• Single-molecule FRET: Using fluorescently labeled ATPAF1 and ATP synthase subunits, researchers can observe assembly dynamics in real-time at the single-molecule level.

• Cryo-electron tomography: This technique allows visualization of ATP synthase assembly intermediates in their native cellular environment at near-atomic resolution.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): This approach can map the interaction interfaces between ATPAF1 and ATP synthase subunits by measuring the exchange rates of backbone amide hydrogens.

• Time-resolved crosslinking mass spectrometry: By capturing transient interactions during assembly using photoreactive crosslinkers, researchers can identify dynamic changes in the interaction network.

• In-cell NMR spectroscopy: This technique can provide atomic-level insights into ATPAF1 interactions within living cells without artificial purification steps .

How does RNA binding potentially influence ATPAF1 function in ATP synthase assembly?

Recent research has revealed intriguing connections between RNA binding and mitochondrial protein assembly, which may extend to ATPAF1 function:

• RNA co-factor hypothesis: Similar to findings with ATP5A1 , ATPAF1 may interact with specific RNAs that serve as co-factors during the assembly process, potentially stabilizing intermediate complexes or facilitating correct spatial orientation of subunits.

• RNA-facilitated import: RNA binding might influence the import of ATPAF1 or its client proteins into mitochondria, as suggested by research showing RNA promotes mitochondrial import of F1-ATP synthase .

• Regulatory RNA interactions: ATPAF1 may interact with regulatory RNAs that modulate its activity in response to cellular energy demands or stress conditions.

• Experimental approaches to investigate RNA involvement:

  • RNA immunoprecipitation (RIP) using ATPAF1 antibodies to identify interacting RNAs

  • eCLIP-Seq to map RNA binding sites at nucleotide resolution

  • Functional assays comparing ATP synthase assembly efficiency in the presence or absence of candidate RNAs

  • Proximity ligation assays (RNA-PLA) to visualize RNA-protein interactions in situ

• Evolutionary conservation: Analysis of RNA binding domains across species could reveal conserved mechanisms of RNA-assisted assembly of mitochondrial complexes.

• Therapeutic implications: If RNA binding proves essential for ATPAF1 function, it could represent a novel therapeutic target for mitochondrial disorders characterized by ATP synthase assembly defects .

How do polyclonal versus monoclonal ATPAF1 antibodies compare in different research applications?

When selecting between polyclonal and monoclonal ATPAF1 antibodies, researchers should consider their specific experimental requirements, with polyclonals offering sensitivity advantages and monoclonals providing specificity benefits .

What are the considerations when selecting between different manufacturers' ATPAF1 antibodies for specific applications?

When choosing between different manufacturers' ATPAF1 antibodies, researchers should evaluate:

• Immunogen design and coverage:

  • Cusabio's polyclonal antibody uses recombinant Human ATP synthase mitochondrial F1 complex assembly factor 1 protein (amino acids 58-143) as the immunogen

  • Other manufacturers may use different immunogens (full-length protein, specific peptides, or different regions)

  • Select antibodies whose immunogen aligns with your region of interest

• Validation data comprehensiveness:

  • Evaluate the extent of validation data provided (knockout controls, peptide competition, cross-reactivity testing)

  • Consider the relevance of validation to your specific application

  • Check for peer-reviewed publications using the antibody

• Species compatibility with your model system:

  • Some antibodies work across multiple species (mouse/rat/human)

  • Others are human-specific

  • Verify cross-reactivity claims with validation data

• Application-specific optimization:

  • For Western blotting: Check recommended dilutions and documented molecular weight

  • For immunofluorescence: Verify subcellular localization matches expected mitochondrial pattern

  • For ELISA: Compare sensitivity data and dynamic range

• Conjugation options and detection system compatibility:

  • HRP-conjugated antibodies eliminate secondary antibody steps

  • Consider fluorescent conjugates for multiplexing

  • Evaluate whether native or conjugated format is optimal for your application

• Storage buffer composition and stability:

  • Compare preservatives, carrier proteins, and stabilizers

  • Consider compatibility with your experimental system

  • Evaluate claimed shelf-life and stability data

How can researchers distinguish between primary assembly defects and secondary effects when studying ATPAF1 dysfunction?

Distinguishing primary assembly defects from secondary consequences when investigating ATPAF1 dysfunction requires a multi-faceted analytical approach:

• Temporal analysis of assembly intermediates:

  • Perform pulse-chase experiments to track the formation of assembly intermediates over time

  • Compare the kinetics of assembly in normal versus ATPAF1-deficient conditions

  • Primary defects will manifest early in the assembly timeline

• Subunit-specific accumulation patterns:

  • Use antibodies against various ATP synthase subunits to determine which components accumulate

  • Primary ATPAF1 defects typically show specific accumulation of F1-alpha and F1-beta subunits

  • Secondary defects often show more generalized patterns affecting multiple complexes

• Complementation studies:

  • Express wild-type ATPAF1 in deficient cells and assess rescue of assembly

  • Immediate rescue suggests a primary role for ATPAF1

  • Delayed or partial rescue may indicate secondary adaptations

• Multi-complex analysis:

  • Assess other mitochondrial complexes (I-IV) alongside ATP synthase

  • Primary ATPAF1 defects should predominantly affect ATP synthase

  • Secondary mitochondrial dysfunction affects multiple complexes

• Isolation of assembly intermediates:

  • Use gradient centrifugation or native electrophoresis to isolate assembly intermediates

  • Characterize these intermediates by mass spectrometry

  • Compare the composition to predicted patterns for specific assembly factor defects

• Mitochondrial stress markers:

  • Evaluate markers of mitochondrial stress (e.g., mtHSP70 upregulation, PINK1 accumulation)

  • Primary assembly defects may show specific stress signatures

  • Secondary effects typically show broader stress responses

What analytical methods should be used to quantify changes in ATPAF1 expression and localization under stress conditions?

To accurately quantify ATPAF1 expression and localization changes under stress conditions, researchers should employ these analytical methods:

• Quantitative expression analysis:

  • qRT-PCR for mRNA expression changes (using validated reference genes)

  • Quantitative Western blotting with fluorescent secondary antibodies for precise protein quantification

  • ELISA using HRP-conjugated ATPAF1 antibody for high-throughput screening

  • Absolute quantification using recombinant ATPAF1 standards

• Subcellular distribution assessment:

  • Sub-mitochondrial fractionation (matrix, inner membrane, intermembrane space)

  • Super-resolution microscopy to track ATPAF1 localization within mitochondria

  • Proximity ligation assays to quantify associations with different mitochondrial compartment markers

  • Protease protection assays to determine membrane integration state

• Dynamic localization monitoring:

  • Live-cell imaging using fluorescently tagged ATPAF1 (validating that tagging doesn't impair function)

  • FRAP (Fluorescence Recovery After Photobleaching) to measure mobility

  • Correlative light and electron microscopy for ultrastructural context

  • Split-GFP complementation to visualize interaction with partner proteins

• Mathematical modeling approaches:

  • Bayesian statistical methods for integrating multiple data types

  • Machine learning algorithms for pattern recognition in complex datasets

  • Principal component analysis to identify key variables in stress responses

  • Hierarchical clustering to identify condition-specific expression patterns

• Validation standards:

  • Include multiple time points to capture temporal dynamics

  • Use several distinct stress conditions to identify specific versus general responses

  • Compare results across cell types to identify cell-specific versus universal responses

  • Include dose-response analyses for chemical stressors

How can researchers integrate ATPAF1 data with other mitochondrial biomarkers to develop comprehensive profiles of mitochondrial dysfunction?

Developing comprehensive mitochondrial dysfunction profiles through ATPAF1 data integration requires sophisticated multi-parametric approaches:

• Multi-omics integration framework:

  • Combine proteomics data on ATPAF1 and ATP synthase components

  • Integrate transcriptomics to identify coordinated gene expression patterns

  • Incorporate metabolomics to connect assembly defects with metabolic consequences

  • Add mitochondrial genomics to assess mtDNA stability and copy number

• Functional-structural correlation:

  • Pair ATPAF1 expression/localization data with ATP synthesis measurements

  • Correlate with respiratory chain complex activity assays

  • Link to mitochondrial membrane potential measurements

  • Connect with mitochondrial morphology parameters (fission/fusion balance)

• Temporal profiling approach:

  • Create time-course experiments capturing early to late dysfunction markers

  • Map the sequence of events following ATPAF1 perturbation

  • Identify critical transition points between adaptive and maladaptive responses

  • Determine reversibility windows for different dysfunction parameters

• Machine learning classification models:

  • Develop supervised learning algorithms to classify dysfunction patterns

  • Create unsupervised clustering to identify novel dysfunction signatures

  • Implement feature importance analysis to prioritize key biomarkers

  • Design predictive models for progression of mitochondrial dysfunction

• Visualization and analysis tools:

  • Create multi-parameter radar plots for dysfunction signatures

  • Develop network analysis visualizations for protein-protein interactions

  • Implement hierarchical clustering heatmaps for pattern recognition

  • Design Sankey diagrams to trace metabolic flux alterations

• Clinical correlation framework:

  • Link laboratory parameters to clinical manifestations

  • Correlate ATPAF1 dysfunction signatures with disease progression

  • Associate with treatment response markers

  • Connect with patient-reported outcomes for clinical relevance

By implementing these comprehensive analytical approaches, researchers can develop nuanced profiles of mitochondrial dysfunction that extend beyond simple binary classifications, enabling personalized approaches to mitochondrial disease diagnosis and treatment.