CYP27A1 Antibody

What is CYP27A1 and why is it important in research?

CYP27A1 is a mitochondrial P-450 enzyme with broad substrate specificity for C27 sterols, including cholesterol and 7-ketocholesterol. The enzyme plays a crucial role in cholesterol metabolism and bile acid synthesis, catalyzing the conversion of cholesterol to bile acids, thereby regulating cholesterol homeostasis. CYP27A1 is particularly important in research because mutations in this gene cause cerebrotendinous xanthomatosis, a rare autosomal recessive lipid storage disease . Additionally, CYP27A1 expression levels have been correlated with prognosis in certain cancers, making it a valuable research target in oncology . Its broad tissue distribution and role in modifying the biological activity of oxidized sterols also make it relevant for research in ophthalmology, neurology, and metabolism.

What are the most common forms of CYP27A1 antibodies available for research?

Commercial CYP27A1 antibodies are available in multiple formats to accommodate diverse experimental requirements. The most widely used is the mouse monoclonal IgG2a kappa light chain antibody (such as CYP27A1 Antibody G-2) that detects CYP27A1 of mouse, rat, and human origin . These antibodies come in:

• Non-conjugated form for standard applications

• Conjugated formats including:

  • Agarose conjugates for immunoprecipitation

  • Horseradish peroxidase (HRP) conjugates for enhanced western blotting sensitivity

  • Fluorescent conjugates including phycoerythrin (PE), fluorescein isothiocyanate (FITC), and multiple Alexa Fluor® variants for immunofluorescence and flow cytometry applications

The choice depends on the specific experimental methodology, with consideration for detection method, signal amplification requirements, and multiplexing needs.

How can I validate the specificity of my CYP27A1 antibody?

Validating antibody specificity is a critical step in ensuring reliable research outcomes. For CYP27A1 antibodies, a comprehensive validation approach includes:

• Positive controls: Use tissues or cell lines known to express CYP27A1 (e.g., liver tissue, ARPE19 cells which show a single band at 47 kDa)

• Knockout/knockdown validation: Compare antibody reactivity in wild-type cells versus CYP27A1 knockout (generated via CRISPR/Cas9) or knockdown (via siRNA) cells

• Multiple detection methods: Confirm specificity across different techniques (western blot, immunofluorescence, ELISA) to ensure consistent results

• Cross-reactivity assessment: Test against related cytochrome P450 family members to ensure specificity

• Peptide competition: Pre-incubate the antibody with a specific blocking peptide to confirm signal suppression

• Comparison with mRNA expression: Correlate protein detection with mRNA levels using techniques like RNAscope to verify concordance (noting that intratumoral concordance between protein and mRNA has been observed at approximately 77.7%)

What are the optimal conditions for western blotting using CYP27A1 antibodies?

Western blotting with CYP27A1 antibodies requires optimized conditions to detect the approximately 47 kDa protein. Based on published protocols:

• Sample preparation:

  • For tissue samples, mitochondrial enrichment improves detection since CYP27A1 is primarily localized to mitochondria

  • Cell fractionation may reveal different expression patterns (e.g., in neural retina, CYP27A1 appears as a high molecular weight smear in soluble fractions, while in RPE/choroid fractions it appears as a dimer)

• Antibody dilutions:

  • Primary antibody: Typically used at 1:2000 dilution (though this varies by manufacturer and application)

  • Secondary antibody: HRP-conjugated anti-mouse IgG at 1:5000-1:55,000 depending on detection system sensitivity

• Visualization systems:

  • Enhanced chemiluminescence (ECL) works well for standard applications

  • For stronger signal amplification, consider using signal enhancers or directly conjugated HRP antibodies

• Controls:

  • Include both positive controls (liver tissue) and negative controls (CYP27A1 knockout cells if available)

  • β-actin or other housekeeping proteins are essential for normalization

What methodology should I use for immunolocalization of CYP27A1 in tissue sections?

Effective immunolocalization of CYP27A1 requires careful consideration of fixation, antigen retrieval, and detection methods:

• Tissue preparation:

  • Fixation in 4% paraformaldehyde for 4 hours preserves epitope accessibility

  • For retinal tissues, 100 μm vibratome sections have been successfully used

• Antibody protocol:

  • Primary antibody: Anti-human CYP27A1 at 1:250 dilution

  • Secondary antibody: Fluorophore-conjugated (e.g., Cy5) goat anti-rabbit at appropriate dilution

  • Nuclear counterstain (e.g., DAPI) aids in cellular localization

• Imaging:

  • Confocal microscopy provides superior resolution for subcellular localization

  • Z-stack imaging helps confirm mitochondrial localization patterns

• Expected patterns:

  • In retinal tissue: Strong signal in photoreceptor inner segments with fainter expression in Müller cells, RPE, and choroid

  • In liver: Predominantly hepatocellular with mitochondrial pattern

  • In breast cancer tissue: Variable expression correlating with prognostic features

How can CYP27A1 antibodies be used in studying protein-protein interactions?

CYP27A1 antibodies can elucidate protein interaction networks through several methodologies:

• Co-immunoprecipitation (Co-IP):

  • Use agarose-conjugated CYP27A1 antibodies (e.g., sc-390974 AC) to pull down CYP27A1 and associated proteins

  • Identify binding partners through mass spectrometry or western blotting

  • Verify interactions through reverse Co-IP (using antibodies against putative binding partners)

• Proximity ligation assay (PLA):

  • Detects protein interactions in situ with single-molecule sensitivity

  • Requires specific primary antibodies against both CYP27A1 and potential interacting proteins

  • Provides spatial information about interaction sites within cells

• Chromatin immunoprecipitation (ChIP):

  • For studying CYP27A1's potential interactions with DNA or chromatin-associated proteins

  • Typically requires cross-linking followed by immunoprecipitation with specific antibodies

• Bimolecular fluorescence complementation (BiFC):

  • Fusion proteins containing CYP27A1 and potential partners linked to complementary fragments of fluorescent proteins

  • Interaction reconstitutes fluorescence, visualizable by microscopy

These approaches have revealed interactions between CYP27A1 and mitochondrial proteins, potentially explaining its role in compound sensitivity mechanisms observed in cancer cells .

How does CYP27A1 expression vary across different tissues and what detection methods are most appropriate?

CYP27A1 displays distinct tissue-specific expression patterns that require tailored detection approaches:

For tissues with low expression, more sensitive detection methods are recommended:

• RNAscope for mRNA detection with cellular resolution

• Signal amplification systems for protein detection

• Tyramide signal amplification (TSA) for immunohistochemistry

• Mass spectrometry-based proteomics for absolute quantification

What is the relationship between CYP27A1 mRNA and protein expression levels in research contexts?

The relationship between CYP27A1 mRNA and protein expression shows moderate but imperfect correlation, with important research implications:

• Concordance levels:

  • Studies in breast cancer tissue have shown approximately 77.7% intratumoral concordance (r = 0.43) between CYP27A1 protein and mRNA expression

  • 62.7% of tumors showed concordant low expression

  • 15% showed concordant high expression

  • 8.3% had low protein/high mRNA expression

  • 14% had high protein/low mRNA expression

• Prognostic significance:

  • Combined high CYP27A1 mRNA and protein expression (mRNA+/protein+) was significantly associated with longer survival compared to double negative expression

  • mRNA expression alone (mRNA+/protein−) showed similar but weaker statistical significance

  • Protein expression alone (mRNA−/protein+) trended toward longer event-free survival but without statistical significance

• Research recommendations:

  • Assess both mRNA (by qPCR or RNAscope) and protein (by IHC or western blot) when possible

  • Consider post-transcriptional regulation mechanisms when interpreting discordant results

  • For prognostic studies, combined assessment provides the most robust predictive value

How can I design experiments to study the enzymatic activity of CYP27A1 in conjunction with antibody-based detection?

Comprehensive analysis of CYP27A1 function requires combining antibody detection with enzymatic activity measurements:

• Enzyme activity assays:

  • Measure conversion of cholesterol to 27-hydroxycholesterol using LC-MS/MS

  • Assess 7-ketocholesterol hydroxylation to 27OH7kCh, which renders the compound non-toxic to cells

  • Correlate enzymatic activity with protein levels detected by antibodies

• Structure-function experimental design:

  • Use site-directed mutagenesis to create CYP27A1 variants

  • Detect mutant proteins with antibodies to confirm expression

  • Measure enzymatic activity to establish structure-function relationships

  • Connect to physiological outcomes (e.g., cell toxicity assays)

• Pharmacological inhibition:

  • Test CYP27A1 inhibitors and correlate with antibody-detected protein levels

  • Use natural compounds like limonoids that have shown CYP27A1-dependent activity

  • Perform rescue experiments with wild-type CYP27A1 overexpression

• In situ activity measurement:

  • Combine immunofluorescence with fluorescent substrate analogs

  • Use mitotracker dyes to confirm mitochondrial localization

  • Correlate localization with enzymatic function

How can CYP27A1 antibodies be used to study its role in cancer progression and prognosis?

CYP27A1 antibodies have proven valuable in cancer research, particularly for prognostic assessment:

• Immunohistochemical analysis in tumor samples:

  • Standard IHC protocols with anti-CYP27A1 antibodies provide prognostic information

  • Inter-reader agreement for dichotomized CYP27A1 expression scoring is high (kappa = 0.93 for IHC)

  • Correlation with clinicopathological features reveals that high CYP27A1 expression is associated with:

    • Adverse tumor characteristics such as higher histological grade

    • High Ki67 expression

    • Yet paradoxically, improved recurrence-free survival

• Prognostic assessment workflow:

  • Determine cut-off values for high versus low expression

  • Score tissue microarrays or whole sections

  • Correlate with clinical data using multivariate Cox proportional hazards models

  • Consider stratification by hormone receptor status (particularly important in breast cancer)

• Cancer type-specific considerations:

  • In breast cancer: High CYP27A1 expression is a favorable prognostic marker in premenopausal patients with lymph node-negative disease

  • In ER+ tumors: Hazard ratio for high CYP27A1 protein expression is 0.20 (95% CI: 0.06-0.8), indicating strong protective effect

  • In ER- tumors: No significant prognostic impact (HR: 1.04, 95% CI: 0.36-3.00)

What methodological approaches can resolve contradictory findings regarding CYP27A1 expression in different disease contexts?

Resolving contradictions in CYP27A1 research requires rigorous methodological approaches:

• Standardization of detection methods:

  • Use validated antibodies with known specificity

  • Employ consistent scoring systems and cut-off values

  • Include positive and negative controls in each experiment

  • Consider both protein and mRNA detection for comprehensive assessment

• Multi-cohort validation:

  • Test hypotheses across multiple independent patient cohorts

  • Stratify by relevant clinical variables (age, hormone receptor status, etc.)

  • Perform meta-analyses when appropriate

• Mechanistic studies to explain contradictions:

  • Investigate context-dependent functions of CYP27A1

  • Consider the impact of substrate availability in different tissues

  • Examine the effects of different metabolites produced by CYP27A1 in different cellular contexts

• Integrated multi-omics approach:

  • Combine protein, mRNA, metabolite, and clinical data

  • Use machine learning to identify patterns that may explain contradictory findings

  • Develop predictive models that incorporate multiple biomarkers

How can CYP27A1 antibodies be applied in drug sensitivity and resistance studies?

CYP27A1 antibodies serve as crucial tools in pharmacological research:

• Correlation with drug sensitivity:

  • CYP27A1 expression levels correlate with sensitivity to certain compounds (e.g., haperforin G, harrpernoid D)

  • Higher CYP27A1 expression is associated with increased sensitivity (lower IC50 values)

• Mechanistic validation through genetic approaches:

  • Use CYP27A1 antibodies to confirm knockdown efficiency after siRNA treatment

  • Verify complete protein loss in CRISPR/Cas9-generated CYP27A1 knockout cell lines

  • Confirm restoration of expression in complementation experiments

• Prediction of drug response:

  • Develop IHC-based scoring systems to predict patient response to therapies

  • Create decision trees incorporating CYP27A1 expression with other biomarkers

  • Validate in clinical sample sets with known treatment outcomes

• Target engagement studies:

  • Use competitive binding assays between drugs and antibodies

  • Perform drug affinity responsive target stability (DARTS) assays with antibody detection

  • Employ cellular thermal shift assays (CETSA) followed by western blotting with CYP27A1 antibodies

How can multiplexed imaging with CYP27A1 antibodies advance our understanding of its cellular interactions?

Multiplexed imaging techniques enhance our understanding of CYP27A1's role in cellular processes:

• Multiplex immunofluorescence approaches:

  • Combine CYP27A1 antibodies with markers for organelles (particularly mitochondria)

  • Use spectral unmixing to resolve closely related fluorophores

  • Apply cyclic immunofluorescence for high-parameter imaging (>10 markers)

  • Employ CODEX or other spatial proteomics platforms for comprehensive analysis

• Co-localization analysis methodologies:

  • Calculate Mander's overlap coefficient or Pearson's correlation for quantitative assessment

  • Use super-resolution microscopy (STORM, PALM, STED) to resolve sub-mitochondrial localization

  • Perform live-cell imaging with fluorescently tagged CYP27A1 to track dynamic interactions

• Spatial context assessment:

  • Examine CYP27A1 distribution in relation to tissue architecture

  • Analyze expression gradients in specialized tissues like retina

  • Map distribution relative to pathological features in disease tissues

What are the challenges in developing and validating monoclonal versus polyclonal CYP27A1 antibodies for specific research applications?

Different antibody types present distinct advantages and challenges for CYP27A1 research:

• Monoclonal antibodies (e.g., CYP27A1 Antibody G-2):

  • Advantages: Consistent specificity across batches, reduced background, superior for quantitative applications

  • Challenges: May recognize limited epitopes, potentially affected by post-translational modifications, sometimes less sensitive

  • Validation requirements: Epitope mapping, cross-reactivity testing, knockout validation

  • Best applications: Quantitative western blotting, standardized immunohistochemistry, flow cytometry

• Polyclonal antibodies:

  • Advantages: Recognize multiple epitopes, potentially more robust to protein denaturation, often higher sensitivity

  • Challenges: Batch-to-batch variability, potential for cross-reactivity, higher background

  • Validation requirements: Extensive specificity testing across applications, pre-absorption controls

  • Best applications: Immunoprecipitation, detection of low-abundance targets, certain immunofluorescence applications

• Application-specific considerations:

  • For quantitative studies: Monoclonal antibodies provide more reliable measurements

  • For detection of modified CYP27A1: Epitope-specific antibodies may be required

  • For evolutionary studies across species: Consider epitope conservation when selecting antibodies

How can advanced proteomics approaches incorporating CYP27A1 antibodies enhance biomarker discovery?

Integration of CYP27A1 antibodies with proteomics offers powerful biomarker discovery capabilities:

• Immunoprecipitation coupled to mass spectrometry (IP-MS):

  • Enrich CYP27A1 and interacting proteins using specific antibodies

  • Identify novel binding partners through unbiased MS analysis

  • Compare interaction networks between normal and disease states

  • Validate findings with targeted approaches (Co-IP, PLA)

• Antibody-based enrichment for post-translational modification analysis:

  • Immunoprecipitate CYP27A1 followed by MS analysis of modifications

  • Identify regulatory PTMs that might affect enzymatic activity

  • Develop modification-specific antibodies for high-throughput screening

• Reverse phase protein arrays (RPPA):

  • Use validated CYP27A1 antibodies in high-throughput protein quantification

  • Screen large sample cohorts for expression patterns

  • Correlate with clinical outcomes or drug responses

  • Integrate with other biomarker data for comprehensive analysis

• Activity-based protein profiling:

  • Apply methods like isoTOP-ABPP (isotopic tandem orthogonal proteolysis-activity-based protein profiling)

  • Combine with CYP27A1 antibodies for target validation

  • Identify protein activity changes in response to compounds or disease states