APOOL Antibody

What is APOOL and what cellular functions does it perform?

APOOL (apolipoprotein O-like) is a component of the MICOS complex, a large protein complex located in the mitochondrial inner membrane. It plays crucial roles in maintaining crista junctions, inner membrane architecture, and forming contact sites with the outer membrane. APOOL specifically binds to cardiolipin (in vitro) but not to the precursor lipid phosphatidylglycerol, highlighting its selectivity for specific mitochondrial lipids . Its full name is apolipoprotein O-like, and it was formerly known by several other identifiers including CXorf33, FAM121A, and MGC129748 .

The protein has a calculated molecular weight of 29 kDa (268 amino acids) and is typically observed at approximately 30 kDa in experimental conditions . Its function in mitochondrial architecture makes it a significant target for studies investigating mitochondrial dynamics and related pathologies.

APOOL antibodies from multiple manufacturers have been confirmed to react with samples from the following species:

• Human

• Mouse

• Rat

This cross-species reactivity has been validated in various sample types including human cell lines (HeLa, LNCaP, HepG2), mouse heart tissue, rat heart tissue, human kidney tissue, and human placenta tissue . The consistent reactivity across species suggests conservation of the APOOL protein structure and epitopes among these mammals, making these antibodies versatile tools for comparative studies.

How should I store and handle APOOL antibodies for optimal performance?

Proper storage and handling of APOOL antibodies is critical for maintaining their reactivity and specificity. Based on manufacturer recommendations, the following guidelines should be followed:

• Storage temperature: Store at -20°C for long-term storage. The antibodies are typically stable for one year after shipment when stored properly .

• Short-term storage: For frequent use within a month, storage at 4°C is acceptable .

• Buffer composition: Most APOOL antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 .

• Aliquoting: While some manufacturers indicate that aliquoting is unnecessary for -20°C storage , it is generally recommended to aliquot antibodies to avoid repeated freeze-thaw cycles which can degrade antibody quality .

• Handling precautions: Due to the presence of sodium azide in the storage buffer, appropriate safety precautions should be followed when handling these reagents.

How can I validate the specificity of APOOL antibodies in my experimental system?

Validating antibody specificity is critical for reliable experimental results. For APOOL antibodies, consider implementing the following validation strategies:

• Knockdown/knockout controls: Several APOOL antibodies are knockdown validated . Generate APOOL knockdown or knockout cell lines using siRNA or CRISPR-Cas9 and compare antibody signal between wild-type and depleted samples.

• Multiple antibody approach: Use antibodies from different vendors or those targeting different epitopes of APOOL. Concordant results increase confidence in specificity.

• Tissue/cell type validation: APOOL antibodies show positive signals in specific tissues and cell types. For Western blot, validated samples include HeLa cells, LNCaP cells, HepG2 cells, mouse heart tissue, and rat heart tissue . For IHC, human kidney and placenta tissues have been validated .

• Blocking peptide experiments: Use a specific blocking peptide corresponding to the immunogen used to generate the antibody. Competitive binding should reduce or eliminate specific signals.

• Molecular weight verification: Confirm that the observed molecular weight matches the expected size of APOOL (calculated as 29 kDa, typically observed at 30 kDa) .

• Cross-reactivity assessment: Text mining methods can extract information about antibody specificity issues from literature to identify potentially problematic antibodies .

What are the optimal conditions for immunohistochemistry with APOOL antibodies?

Successful immunohistochemistry with APOOL antibodies requires attention to several technical parameters:

• Antigen retrieval: For paraffin-embedded tissues, antigen retrieval with TE buffer at pH 9.0 is suggested. Alternatively, citrate buffer at pH 6.0 can be used .

• Antibody dilution: For IHC applications, a dilution range of 1:150-1:600 is recommended . The optimal dilution should be determined empirically for each experimental system.

• Positive control tissues: Human kidney tissue and human placenta tissue have been validated as positive controls for APOOL antibody in IHC applications .

• Detection system: The specific detection system (e.g., HRP-DAB, fluorescence) should be optimized based on the experimental goals and the antibody's isotype (typically rabbit IgG for APOOL antibodies) .

• Fixation conditions: Standard formalin fixation and paraffin embedding procedures are compatible with APOOL detection, but optimization may be required for other fixation methods.

• Incubation parameters: Time, temperature, and washing steps should be optimized for the specific antibody and detection system used.

How can I predict and mitigate potential cross-reactivity of APOOL antibodies?

Antibody cross-reactivity can lead to misleading results. For APOOL antibody research, consider these approaches:

• Probability-based assessment: The probability of cross-reactivity between polypeptides depends on shared epitopes. Mathematical models can help predict the likelihood of cross-reactions when amino acid compositions are known .

• Epitope analysis: Examine the immunogen sequence used to generate the antibody. For example, one APOOL antibody was developed against a recombinant protein corresponding to amino acids: "ATLGATVCYPVQSVIIAKVTAKKVYATSQQIFGAVKSLWTKSSKEESLPKPKEKTKLGSSSEIEVPAKTTHVLKHSVPLPTELSSEAKTKSESTSGATQFMPDPKLMDHGQSHPED" . BLAST or align this sequence against other proteins to identify potential cross-reactants.

• Multi-parameter validation: Use orthogonal techniques (WB, IHC, IF) to confirm specificity. Concordant results across different methods increase confidence in antibody specificity.

• Negative controls: Include appropriate negative controls such as isotype controls, no-primary-antibody controls, and tissues/cells known not to express APOOL.

• Competition assays: Pre-incubate the antibody with purified APOOL protein or the immunizing peptide to demonstrate signal specificity.

• Data mining approach: Leverage public databases of antibody specificity problems to identify known cross-reactivity issues with similar antibodies .

How can data mining approaches enhance APOOL antibody research?

Data mining is increasingly important for advanced antibody research. For APOOL antibody investigations:

• Sequence-structure-function relationships: Mining of public and proprietary antibody data can accelerate discovery by identifying optimal properties for therapeutic or research antibodies .

• Observed Antibody Space: The OAS database contains over half a billion naturally occurring antibody sequences across diverse immune states and individuals. Comparing engineered sequences with naturally occurring ones could help identify modifications that reduce immunogenicity or improve other properties .

• Patent mining: Analysis of patent databases containing antibody sequences (like the study from Krawczyk et al. analyzing over 245,000 antibody sequences from 16,526 patent families) can provide insights into successful antibody designs .

• Experimental validation of mining-derived insights: High-throughput mutational studies can systematically evaluate property changes upon introducing modifications identified through global data mining, strengthening computational sequence-activity models .

• Text mining for reliability assessment: Text mining methods can extract statements about antibody specificity issues from literature to construct knowledge bases alerting users about potentially problematic antibodies .

What is the recommended protocol for Western blot using APOOL antibodies?

For optimal Western blot results with APOOL antibodies, follow this methodological approach:

• Sample preparation:

  • Extract proteins from validated sample types (HeLa cells, LNCaP cells, HepG2 cells, mouse heart tissue, rat heart tissue)

  • Use appropriate lysis buffer containing protease inhibitors

  • Determine protein concentration for equal loading

• Gel electrophoresis:

  • Load 20-40 μg protein per lane

  • Use 10-12% SDS-PAGE gels for optimal separation of the 30 kDa APOOL protein

• Transfer:

  • Transfer to PVDF or nitrocellulose membrane using standard protocols

  • Verify transfer efficiency with reversible staining (Ponceau S)

• Blocking:

  • Block with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature

• Primary antibody incubation:

  • Dilute APOOL antibody 1:1000-1:6000 in blocking solution

  • Incubate overnight at 4°C with gentle agitation

• Washing and secondary antibody:

  • Wash 3x with TBST

  • Incubate with appropriate HRP-conjugated secondary antibody (anti-rabbit IgG for most APOOL antibodies)

  • Dilute secondary antibody according to manufacturer's recommendations

• Detection:

  • Use enhanced chemiluminescence (ECL) detection

  • Expected molecular weight: 30 kDa

• Controls:

  • Include positive control samples known to express APOOL

  • Consider loading controls (β-actin, GAPDH) for normalization

How can I optimize immunofluorescence experiments with APOOL antibodies?

For high-quality immunofluorescence results with APOOL antibodies:

• Cell preparation:

  • Culture cells on glass coverslips or chamber slides

  • HeLa cells are validated for positive IF/ICC detection

• Fixation and permeabilization:

  • Fix cells with 4% paraformaldehyde for 10-15 minutes at room temperature

  • Permeabilize with 0.1-0.3% Triton X-100 in PBS for 5-10 minutes

• Blocking:

  • Block with 1-5% BSA in PBS for 30-60 minutes at room temperature

• Primary antibody incubation:

  • Dilute APOOL antibody 1:200-1:800 in blocking solution

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

• Washing and secondary antibody:

  • Wash 3x with PBS

  • Incubate with fluorophore-conjugated secondary antibody (specific to rabbit IgG)

  • Dilute secondary antibody according to manufacturer's recommendations

  • Incubate for 1 hour at room temperature in the dark

• Counterstaining and mounting:

  • Counterstain nuclei with DAPI (1 μg/mL) for 5 minutes

  • Mount with anti-fade mounting medium

• Confocal microscopy:

  • Visualize using appropriate excitation/emission wavelengths

  • APOOL should show mitochondrial localization pattern

  • Perform z-stack imaging for complete cellular visualization

• Controls and co-localization:

  • Include negative controls (no primary antibody)

  • Consider co-staining with mitochondrial markers for co-localization studies

How do I implement robust validation practices for APOOL antibody experiments?

Implementing comprehensive validation practices ensures reliable and reproducible APOOL antibody research:

• Antibody authentication:

  • Verify antibody specificity using multiple techniques (WB, IHC, IF)

  • Confirm correct molecular weight (30 kDa) in Western blot

  • Document antibody information: catalog number, lot number, RRID (e.g., AB_3086061)

• Positive and negative controls:

  • Include validated positive controls (HeLa cells, LNCaP cells, HepG2 cells for WB; human kidney and placenta tissues for IHC)

  • Implement appropriate negative controls (samples lacking APOOL expression)

  • Use siRNA/shRNA knockdown or CRISPR knockout samples as specificity controls

• Reproducibility assessment:

  • Perform at least three independent biological replicates

  • Document all experimental conditions thoroughly

  • Consider using multiple antibody clones targeting different epitopes

• Quantification and statistical analysis:

  • Use appropriate software for signal quantification

  • Apply statistical tests to determine significance of findings

  • Present data with error bars and p-values

• Enhanced validation approaches:

  • Genetic strategies: test on knockout/knockdown samples

  • Independent antibody validation: use antibodies from multiple vendors

  • Orthogonal validation: confirm findings with non-antibody methods

• Documentation and reporting:

  • Follow guidelines for antibody reporting in publications

  • Include all relevant antibody information (source, catalog number, RRID)

  • Describe all validation steps performed

What are the best practices for designing experiments to detect APOOL in different cellular compartments?

APOOL is primarily localized to the mitochondrial inner membrane as part of the MICOS complex. To effectively detect and study its cellular distribution:

• Subcellular fractionation approach:

  • Perform mitochondrial isolation using standardized protocols

  • Further separate mitochondrial outer membrane, inner membrane, and matrix fractions

  • Analyze APOOL content in each fraction by Western blot

  • Include fraction-specific markers (e.g., TOM20 for outer membrane, COX IV for inner membrane)

• Super-resolution microscopy:

  • Use techniques like STED, STORM, or PALM for nanoscale resolution

  • Co-stain with established mitochondrial markers

  • Perform 3D reconstruction to visualize APOOL distribution within mitochondria

• Proximity labeling methods:

  • Use BioID or APEX2 fusion constructs with APOOL

  • Identify proximal proteins to map the local environment

  • Confirm mitochondrial localization and identify interacting partners

• Live-cell imaging:

  • Generate fluorescent protein-tagged APOOL constructs

  • Validate that tagging doesn't disrupt localization or function

  • Perform time-lapse imaging to study dynamics

• Electron microscopy (EM):

  • Use immuno-EM with gold-labeled secondary antibodies

  • Look for specific labeling at cristae junctions

  • Compare with known MICOS complex components

• Biochemical verification:

  • Perform co-immunoprecipitation with other MICOS components

  • Verify APOOL's cardiolipin binding properties using liposome binding assays

  • Assess the impact of APOOL depletion on mitochondrial ultrastructure