CYP11A1 Antibody

What is CYP11A1 and what role does it play in steroidogenesis?

CYP11A1 (Cytochrome P450 family 11 subfamily A member 1) is a mitochondrial enzyme that catalyzes the first and rate-limiting step in steroid hormone biosynthesis. This 521-amino acid residue protein converts cholesterol to pregnenolone through side-chain hydroxylation and cleavage, which serves as the precursor for all steroid hormones . The enzyme is also known by several aliases including P450SCC (side-chain cleavage), CYPXIA1, and cholesterol 20-22 desmolase . CYP11A1 is essential for the synthesis of all steroid hormones, making it a critical enzyme in endocrine research.

In which tissues is CYP11A1 predominantly expressed?

CYP11A1 is primarily expressed in classical steroidogenic tissues including the adrenal gland, testis, and placenta . In these tissues, it facilitates the production of steroid hormones essential for various physiological functions. Immunohistochemical analyses consistently demonstrate strong CYP11A1 expression in adrenal cortical cells, Leydig cells of the testis, and syncytiotrophoblasts of the placenta . Interestingly, research has also identified CYP11A1 expression in non-classical steroidogenic tissues, including certain immune cells such as CD8+ T cells under specific stimulation conditions .

What are the most common applications for CYP11A1 antibodies in research?

CYP11A1 antibodies are utilized across multiple experimental platforms for antigen-specific immunodetection. The most common applications include:

• Western Blotting (WB): Used to detect CYP11A1 protein expression in tissue lysates, with optimal dilutions ranging from 1:1,000 to 1:10,000 depending on the antibody

• Immunohistochemistry (IHC): For localization of CYP11A1 in tissue sections, particularly effective in paraffin-embedded tissues of steroidogenic organs

• Immunofluorescence (IF): For subcellular localization studies, particularly useful for confirming mitochondrial localization

• ELISA: For quantitative assessment of CYP11A1 levels in biological samples

• Immunocytochemistry (ICC): For cellular localization in cultured cells

How can CYP11A1 enzymatic activity be measured in experimental settings?

Measuring CYP11A1 enzymatic activity, rather than simply detecting protein expression, provides crucial functional data. The most reliable methodological approaches include:

• Pregnenolone ELISA: Quantifying pregnenolone levels in cell culture supernatants directly correlates with CYP11A1 enzymatic activity. This method has been successfully used to demonstrate the effects of CYP11A1 inhibitors such as aminoglutethimide (AMG) .

• Liquid Chromatography-Mass Spectrometry (LC-MS): This highly sensitive technique provides precise quantification of steroid hormones downstream of CYP11A1 activity. LC-MS can detect complete inhibition of steroid hormone biosynthesis following CYP11A1 inhibition .

• Radioactive substrate conversion assays: Using radiolabeled cholesterol as a substrate and measuring the conversion to pregnenolone through scintillation counting or autoradiography.

Researchers should include appropriate controls when assessing enzymatic activity, including positive controls (tissues known to express active CYP11A1) and negative controls (tissues or conditions where CYP11A1 is absent or inhibited).

What strategies should be employed to validate CYP11A1 antibody specificity?

Ensuring antibody specificity is critical for reliable experimental results. Comprehensive validation strategies include:

• Multiple antibody comparison: Use at least two different antibodies targeting distinct epitopes of CYP11A1 to confirm consistent results .

• Peptide competition assays: Pre-incubate the antibody with the immunizing peptide. As demonstrated with anti-CYP11A1 antibody evaluation, specific binding should show at least ten thousand-fold (10,000X) higher affinity to the target CYP11A1 peptide compared to non-specific control peptides .

• Genetic validation: Employ shRNA knockdown of CYP11A1 expression. For example, transfection with Cyp11a1-specific shRNA has been shown to reduce the percentage of Cyp11a1-positive cells from 67.8% to 28.8%, providing confirmation of antibody specificity .

• Positive and negative tissue controls: Test antibodies on tissues known to express CYP11A1 (adrenal gland, testis, placenta) and tissues that don't express the protein .

• Western blot molecular weight verification: Confirm that the detected band corresponds to the expected molecular weight of approximately 60.1 kilodaltons .

How do fixation and sample preparation protocols affect CYP11A1 immunodetection?

Fixation and sample preparation critically impact CYP11A1 detection quality:

• For immunohistochemistry:

  • Formalin fixation followed by paraffin embedding preserves tissue architecture while maintaining CYP11A1 antigenicity

  • Antigen retrieval methods (heat-induced in citrate buffer, pH 6.0) may be necessary to unmask epitopes

  • Optimal antibody dilutions for paraffin sections typically range from 1:500 to 1:1,000

• For Western blotting:

  • Appropriate lysis buffers containing protease inhibitors are essential

  • Mitochondrial enrichment protocols can enhance detection sensitivity

  • Denaturation conditions must be optimized as CYP11A1 is a mitochondrial membrane-associated protein

• For immunofluorescence:

  • Paraformaldehyde (4%) fixation for 15-20 minutes typically preserves CYP11A1 antigenicity

  • Permeabilization with 0.1-0.2% Triton X-100 facilitates antibody access to mitochondrial CYP11A1

  • Co-staining with mitochondrial markers confirms proper subcellular localization

What controls should be included when using CYP11A1 antibodies in research?

Robust experimental design requires appropriate controls:

• Positive tissue controls: Human adrenal, testis, and placenta tissue samples have been validated for CYP11A1 expression .

• Negative controls:

  • Antibody omission control

  • Isotype control antibody

  • Non-steroidogenic tissues (when applicable)

• siRNA/shRNA knockdown controls: As demonstrated in CYP11A1 research, transfection with Cyp11a1-specific shRNA resulted in significantly reduced staining compared to scrambled shRNA controls .

• Inhibitor controls: Aminoglutethimide (AMG) has been used as a pharmacological inhibitor of CYP11A1 enzymatic activity while not affecting protein levels, providing an important functional control .

• Recombinant protein standards: Using purified CYP11A1 protein as a standard for Western blotting can help with quantification and validation.

How can researchers troubleshoot non-specific binding with CYP11A1 antibodies?

When encountering non-specific binding, several methodological adjustments can improve specificity:

• Optimize blocking conditions:

  • Extend blocking time (1-2 hours at room temperature or overnight at 4°C)

  • Test different blocking agents (BSA, milk proteins, normal serum)

  • Use commercial blocking solutions formulated for challenging applications

• Adjust antibody conditions:

  • Increase antibody dilution (test serial dilutions from 1:1,000 to 1:10,000)

  • Reduce incubation temperature (4°C overnight versus room temperature)

  • Add 0.1-0.2% Tween-20 to antibody diluent

• Increase washing stringency:

  • Additional washing steps

  • Higher detergent concentration in wash buffers

  • Longer wash durations

• Pre-adsorption with non-specific proteins:

  • Pre-incubate primary antibody with non-target tissue lysates

  • Use commercially available pre-adsorption kits

• Verify tissue preparation:

  • Ensure complete deparaffinization for IHC

  • Optimize antigen retrieval methods

  • Check for endogenous peroxidase or phosphatase activity

How can contradictory results with different CYP11A1 antibody clones be reconciled?

Contradictory results between antibody clones are common challenges in research. Reconciliation strategies include:

• Epitope mapping analysis: Different antibodies may target distinct regions of CYP11A1. Document the epitope specificity of each antibody and consider how protein folding, post-translational modifications, or protein-protein interactions might affect epitope accessibility.

• Validation with functional assays: When antibody results conflict, validate with functional assays such as enzymatic activity measurements or pregnenolone production .

• Correlation with mRNA expression: Quantitative PCR analysis of CYP11A1 mRNA can provide complementary evidence. As shown in research, IL-4 treatment increased both Cyp11a1 mRNA and protein levels in CD8+ T cells .

• Multi-methodological approach: Combine different detection techniques (WB, IHC, IF) to build a more complete picture. For example, research has shown that Western blotting detected CYP11A1 in placenta tissue lysate at dilutions from 1:1,000 to 1:10,000, while immunofluorescence required different optimization .

• Consider species differences: Ensure antibodies are validated for the species being studied, as epitope conservation varies across species.

What factors might influence CYP11A1 expression levels in experimental systems?

Several factors can affect CYP11A1 expression and should be considered when interpreting data:

• Cytokine environment: IL-4 has been shown to significantly increase Cyp11a1 expression in CD8+ T cells compared to IL-2 alone .

• Cell activation status: T cell receptor stimulation with specific antigens (e.g., SIINFEKL peptide) can alter CYP11A1 expression patterns .

• Hormonal influence: Steroid hormones can regulate CYP11A1 through feedback mechanisms.

• Tissue-specific expression patterns: Expression levels vary significantly between classical steroidogenic tissues (adrenal, testis, placenta) and non-classical sites .

• Developmental stage: CYP11A1 expression can vary throughout development.

• Pathological conditions: Disease states, particularly those affecting steroid-producing tissues, can alter CYP11A1 expression.

How is CYP11A1 being targeted in cancer research?

CYP11A1 has emerged as an important target in hormone-dependent cancer research:

• First-in-class inhibitors: ODM-208, a selective, nonsteroidal, oral CYP11A1 inhibitor, has been developed to halt synthesis of all steroid hormones by targeting the first enzymatic step in steroidogenesis .

• Castration-resistant prostate cancer (CRPC): CYP11A1 inhibition with ODM-208 led to rapid, complete, durable, and reversible inhibition of steroid hormone biosynthesis in patients with CRPC .

• Mechanistic approach: Unlike traditional hormone therapies that target hormone-receptor binding, CYP11A1 inhibition prevents the synthesis of all steroid hormones at the source, potentially overcoming resistance mechanisms .

• Clinical implementation: Administration of CYP11A1 inhibitors is feasible with concomitant corticosteroid replacement therapy to supply essential steroids .

• Toxicity profile: Studies in rats and dogs show that toxicity findings related to CYP11A1 inhibition are reversible after discontinuation of treatment .

What role does CYP11A1 play in immune cell function?

Research has uncovered unexpected roles for CYP11A1 in immune system regulation:

• CD8+ T cell phenotype regulation: CYP11A1 controls the phenotypic conversion of CD8+ T cells from IFN-γ to IL-13 production, linking steroidogenesis to allergic differentiation pathways .

• IL-4 induction: IL-4 treatment significantly increases CYP11A1 expression in CD8+ T cells, suggesting cytokine-regulated expression in non-classical steroidogenic tissues .

• Functional impact: Inhibition of CYP11A1 enzymatic activity with aminoglutethimide prevented IL-4-induced conversion of IFN-γ to IL-13-producing cells .

• In vivo significance: Adoptive transfer of aminoglutethimide-treated CD8+ T cells into sensitized and challenged CD8-deficient recipients failed to restore airway hyperresponsiveness and inflammation, demonstrating the physiological relevance of this pathway .

• Translational potential: This research links local steroidogenesis in T cells to allergic disease progression, suggesting potential therapeutic opportunities.

What advanced techniques are enhancing CYP11A1 research?

Several cutting-edge technologies are advancing CYP11A1 research:

• ZooMAb® Recombinant Monoclonal Antibodies: These offer significantly enhanced specificity, affinity, reproducibility, and stability over conventional monoclonals for CYP11A1 detection .

• Preservative-free antibody formulations: New antibody preparations allow for ambient shipping and storage while maintaining activity .

• Virtual screening approaches: Computational methods have facilitated the development of novel CYP11A1 inhibitors through systematic structure-activity relationship optimization .

• Multi-omics integration: Combining proteomics, metabolomics, and transcriptomics provides comprehensive insights into CYP11A1 function and regulation.

• Single-cell analysis: Techniques examining CYP11A1 expression and activity at the single-cell level reveal heterogeneity within seemingly homogeneous cell populations.

What are the most promising research directions for CYP11A1 antibody applications?

Future research directions with significant potential include:

• Non-classical steroidogenic tissues: Expanding our understanding of CYP11A1 expression and function in tissues not traditionally associated with steroidogenesis, such as the immune system .

• Therapeutic targeting: Further development of selective CYP11A1 inhibitors for hormone-dependent cancers and other conditions .

• Biomarker development: Validating CYP11A1 as a diagnostic or prognostic biomarker in adrenal disorders, gonadal dysfunction, and hormone-dependent cancers.

• Developmental biology: Exploring the role of CYP11A1 in embryonic development and developmental disorders affecting steroidogenic tissues.

• Environmental impacts: Investigating how environmental factors and endocrine-disrupting chemicals affect CYP11A1 expression and activity.

• Structural biology: Determining high-resolution structures of CYP11A1 to facilitate better understanding of its catalytic mechanism and enable structure-based drug design.