ATPIF1 Antibody

What is ATPIF1 and what are its primary biological functions?

ATPIF1 (ATP synthase inhibitory factor 1) is a small mitochondrial protein (~12 kDa) that plays a crucial role in regulating mitochondrial ATP synthase activity. It functions as an endogenous F(1)F(o)-ATPase inhibitor that limits ATP depletion when the mitochondrial membrane potential falls below a threshold.

ATPIF1 primarily:

• Binds to the C-terminal region of a β subunit of the F1-ATPase, inhibiting enzyme activity particularly under low pH conditions

• Prevents excessive ATP hydrolysis during metabolic stress

• Maintains cellular energy homeostasis by preventing ATP consumption when F(1)F(o)-ATP synthase acts as an ATP hydrolase

• Regulates heme synthesis in erythroid tissues by modulating mitochondrial pH and redox potential

• Participates in vascular function through reversible binding to the F1F0-ATPase complex on endothelial cell surfaces

What types of ATPIF1 antibodies are available for research applications?

Several types of ATPIF1 antibodies are available for research:

By antibody class:

• Mouse monoclonal antibodies (e.g., A-3 clone, 5E2D7 clone)

• Rabbit polyclonal antibodies

• Recombinant antibodies (e.g., 3E2 clone)

By conjugation status:

• Unconjugated primary antibodies

• Conjugated forms:

  • Agarose-conjugated (for immunoprecipitation)

  • HRP-conjugated (for enhanced western blot detection)

  • Fluorophore-conjugated: FITC, PE, multiple Alexa Fluor® variants

By species reactivity:

• Human-specific

• Multi-species (human/mouse/rat/bovine)

How should ATPIF1 antibodies be stored and handled to maintain optimal activity?

Based on manufacturer recommendations:

Storage conditions:

• Store at -20°C (most common recommendation)

• Stable for one year after shipment when properly stored

• For certain formulations, aliquoting is unnecessary for -20°C storage

Buffer composition:

• Typically provided in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Some preparations include 0.1% BSA for stability

Handling recommendations:

• Avoid repeated freeze-thaw cycles

• Allow antibody to equilibrate to room temperature before opening

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

• Return to storage conditions promptly after use

What applications can ATPIF1 antibodies be reliably used for?

ATPIF1 antibodies have been validated for multiple applications:

What are the recommended dilutions for different applications of ATPIF1 antibodies?

Based on manufacturer recommendations:

It is strongly recommended to titrate antibodies in each experimental system to obtain optimal results, as performance can be sample-dependent .

How can ATPIF1 antibodies be used to investigate ATPIF1-ATP synthase interactions?

ATPIF1 antibodies can be used to study ATPIF1-ATP synthase interactions through several approaches:

Co-immunoprecipitation:

• Immunoprecipitate ATP synthase complex using anti-ATP synthase antibodies

• Detect co-precipitated ATPIF1 by western blotting with anti-ATPIF1 antibodies

• This approach has revealed ATPIF1 binding to ATP synthase under various metabolic states

Proximity Ligation Assay (PLA):

• Use antibodies against ATPIF1 and ATP synthase subunits (e.g., β-F1-ATPase)

• PLA signals indicate close proximity (<40 nm) between proteins

• Quantify as spots/area, spots/nucleus, or percentage of spots/μm³ of mitochondria

• Note: Mitotracker staining may reduce PLA sensitivity

Immunofluorescence co-localization:

• Co-stain with antibodies against ATPIF1 and ATP synthase subunits

• Calculate Pearson's correlation coefficient to quantify co-localization

• Values close to 1 indicate strong co-localization (e.g., Pearson's r = 0.90 ± 0.01 for ATPIF1 with β-F1-ATPase)

How can ATPIF1 antibodies be used to investigate ATPIF1's role in cancer biology?

ATPIF1 is overexpressed in many tumors and functions as a pro-oncogenic protein. Researchers can use ATPIF1 antibodies to:

Expression analysis:

• Analyze ATPIF1 expression levels in different cancer types via IHC or WB

• Compare expression between tumor and adjacent normal tissues

• Correlate expression with clinical parameters and patient outcomes

Mechanism studies:

• Investigate ATPIF1 interaction with ATP synthase OSCP subunit in cancer cells using IP or PLA

• Study the effect of ATPIF1 on ATP synthase oligomerization and PTP (permeability transition pore) formation

• Examine how ATPIF1-ATP synthase interaction affects apoptosis in cancer cells

T cell function in cancer:

• Assess how ATPIF1 expression affects T cell exhaustion in tumor-infiltrating lymphocytes

• Examine the relationship between ATPIF1 levels and CD8+ T cell function (proliferation, IFN-γ secretion)

• Study ATPIF1's influence on metabolic reprogramming (glycolysis vs. OXPHOS) in T cells

Therapeutic potential:

• Monitor changes in ATPIF1 expression during cancer treatment

• Assess ATPIF1 as a biomarker for therapy response

• Evaluate potential of ATPIF1 modulation for enhancing CAR-T cell efficacy

What is known about ATPIF1's role in cardiac pathophysiology, and how can antibodies help investigate this?

ATPIF1 is specifically upregulated in pathological cardiac hypertrophy but not in physiological hypertrophy. ATPIF1 antibodies can be used to:

Expression analysis:

• Detect ATPIF1 upregulation in different models of cardiac hypertrophy:

  • Pressure overload via transverse aortic constriction (TAC)

  • Myocardial infarction (MI)

  • Phenylephrine-induced hypertrophy in cardiomyocytes

Structural studies:

• Investigate the formation of ATP synthase tetramers when bound to ATPIF1

• Study ATPIF1 cross-linking with ATP synthase subunits using techniques like iqPIR (isobaric quantitative Protein Interaction Reporter)

• Analyze ATPIF1-induced structural changes in ATP synthase

Metabolic reprogramming:

• Examine how ATPIF1 upregulation affects the metabolic shift toward increased glycolysis in hypertrophied hearts

• Study ATPIF1's role in reducing oxidative phosphorylation under cardiac stress conditions

How can ATPIF1 antibodies be used to investigate different binding modes of ATPIF1 with ATP synthase?

Recent research suggests ATPIF1 may interact with ATP synthase through multiple binding modes:

Classical inhibitory binding:

• Study ATPIF1 binding to the catalytic F1 domain during ATP hydrolysis

• Examine the pH-dependence of this interaction

• Investigate how this binding mode prevents ATP consumption during metabolic stress

Alternative binding to OSCP:

• Use antibodies to investigate ATPIF1 binding to the ATP synthase OSCP subunit under normal culture conditions

• Study whether this interaction occurs without the requirement for ATP hydrolysis

• Determine how this binding affects ATP synthase function during ATP synthesis

State-dependent interactions:

• Compare ATPIF1-ATP synthase interactions across different metabolic states:

  • State 2 respiration (substrate without ADP)

  • State 3 respiration (substrate with ADP, active ATP synthesis)

  • ATP hydrolysis conditions

• Detect these interactions using immunoprecipitation followed by western blotting

How can the specificity of ATPIF1 antibodies be validated?

Thorough validation of ATPIF1 antibody specificity is crucial:

Genetic validation approaches:

• Test antibody in ATPIF1 knockout cells created via CRISPR-Cas9

• Verify absence of signal in knockout samples by western blot, IF, and IHC

• Compare with wild-type samples showing expected signal pattern

RNA interference validation:

• Test antibody in cells with ATPIF1 knockdown by siRNA or shRNA

• Verify reduction in signal intensity correlating with knockdown efficiency

• Include non-targeting control siRNA/shRNA samples

Recombinant protein controls:

• Use purified recombinant ATPIF1 protein as a positive control

• Verify antibody detection at the expected molecular weight (~12 kDa)

• Perform peptide competition assays to confirm epitope specificity

Subcellular localization verification:

• Confirm mitochondrial localization through co-staining with established mitochondrial markers

• Calculate Pearson's correlation coefficient with mitochondrial markers (values close to 0.9 indicate strong colocalization)

What are common technical challenges when working with ATPIF1 antibodies, and how can they be addressed?

Challenge: Low signal intensity in western blots

• Solution: Optimize protein extraction from mitochondria using specialized buffers

• Load higher amounts of mitochondrial fractions rather than whole cell lysates

• Use more sensitive detection methods (ECL Prime, SuperSignal West Femto)

• Reduce washing stringency or duration

Challenge: High background in immunofluorescence

• Solution: Use longer blocking times (≥1 hour) with 5% BSA or 10% normal serum

• Include 0.1-0.3% Triton X-100 in antibody diluent to reduce non-specific binding

• Use more dilute antibody concentrations

• Consider using specialized mounting media with anti-fade properties

Challenge: Inconsistent immunoprecipitation results

• Solution: Crosslink antibodies to beads to prevent antibody co-elution

• Use gentler lysis conditions to preserve protein-protein interactions

• Include protease inhibitors freshly in all buffers

• Consider using ATPIF1-GFP fusion constructs for initial optimization

Challenge: Interference in PLA assays

• Solution: Avoid Mitotracker staining as it may reduce PLA sensitivity

• Use alternative mitochondrial markers that don't interfere with the PLA reaction

• Optimize primary antibody concentrations specifically for PLA applications

How should results from different ATPIF1 antibodies be compared and reconciled?

When using multiple ATPIF1 antibodies:

Epitope considerations:

• Map the epitope regions recognized by different antibodies

• Consider that antibodies targeting different epitopes may yield different results

• N-terminal vs. C-terminal targeting antibodies may detect different isoforms

Cross-validation approach:

• Use at least two antibodies from different sources targeting different epitopes

• Compare results across multiple experimental techniques (WB, IF, IHC)

• Prioritize antibodies with published validation in similar applications

Isoform awareness:

• Consider that ATPIF1 has up to 3 reported isoforms in humans

• Determine which isoforms each antibody can detect

• Account for isoform-specific expression patterns in different tissues

Rigorous controls:

• Include the same positive and negative controls when comparing antibodies

• Use the same experimental conditions and samples for direct comparisons

• Document differences in sensitivity and specificity between antibodies

How are ATPIF1 antibodies being used in innovative research approaches?

Single-cell techniques:

• Integration with single-cell RNA sequencing (scRNA-seq) to correlate protein expression with transcriptional profiles

• Single-cell western blotting to analyze ATPIF1 expression heterogeneity within populations

• Single-cell imaging to examine subcellular localization differences

Advanced microscopy applications:

• Super-resolution microscopy (STORM, PALM) to visualize ATPIF1 distribution within mitochondria

• Live-cell imaging using genetically encoded tags to monitor ATPIF1 dynamics

• FRET-based approaches to study ATPIF1-ATP synthase interactions in real-time

Clinical research applications:

• Development of tissue microarray analysis for ATPIF1 expression in cancer cohorts

• Integration of ATPIF1 expression data with patient outcomes in biomarker studies

• Analysis of ATPIF1 in liquid biopsies (circulating tumor cells, exosomes)

How can ATPIF1 antibodies contribute to studying the therapeutic potential of ATPIF1 modulation?

ATPIF1 modulation shows therapeutic potential in several contexts:

Cancer immunotherapy:

• Monitoring ATPIF1 levels in T cells during immunotherapy response

• Evaluating ATPIF1 expression as a predictor of immunotherapy efficacy

• Investigating ATPIF1 overexpression as a strategy to enhance CAR-T cell function

Cardiac protection strategies:

• Assessing ATPIF1 modulation as an intervention in pathological cardiac hypertrophy

• Monitoring ATPIF1 expression changes during heart failure progression

• Investigating the effects of cardiac drugs on ATPIF1 expression and function

Metabolic disease applications:

• Studying ATPIF1's role in metabolic reprogramming in obesity and diabetes

• Investigating ATPIF1 modulation as a strategy to improve mitochondrial function

• Examining tissue-specific ATPIF1 expression patterns in metabolic disorders

How can researchers select the most appropriate ATPIF1 antibody for their specific research questions?

Selection criteria should include:

Research question alignment:

• For protein-protein interactions: Choose antibodies validated for IP and PLA

• For localization studies: Select antibodies optimized for IF/IHC with minimal background

• For quantitative analysis: Use antibodies validated for flow cytometry or ELISA

Technical specifications:

• Species reactivity matching experimental model (human, mouse, rat)

• Monoclonal vs. polyclonal (monoclonals for specificity, polyclonals for sensitivity)

• Clone-specific performance in published literature (e.g., applications, citations)

Validated applications:

• Review validation data provided by manufacturers

• Assess published literature using specific antibody clones

• Consider antibodies with knockout validation when available

Experimental controls:

• Ensure appropriate positive and negative controls are available

• Consider generating ATPIF1 knockout or knockdown models for validation

• Plan for orthogonal verification using multiple antibodies or techniques