CYP17A1 Monoclonal Antibody

What is CYP17A1 and what is its biological significance?

CYP17A1, also known as cytochrome P450 family 17 subfamily A member 1, is a critical enzyme in steroidogenesis that catalyzes two key reactions: 17-alpha-hydroxylation and 17,20-lyase reactions. These functions enable the conversion of pregnenolone and progesterone into dehydroepiandrosterone (DHEA) and androstenedione, respectively . The enzyme plays an essential role in sex steroid production, which is vital for sexual development during both fetal growth and puberty . CYP17A1 is primarily expressed in steroidogenic tissues, with significant detection in adrenal and testicular tissues as demonstrated in immunohistochemical analyses .

The calculated molecular weight of CYP17A1 is approximately 57 kDa, though the observed molecular weight in Western blot analyses typically ranges between 48-51 kDa . This discrepancy may be attributed to post-translational modifications or tissue-specific processing of the protein.

How do CYP17A1 monoclonal antibodies differ from polyclonal antibodies?

CYP17A1 monoclonal antibodies, such as the D-12 clone (sc-374244), recognize a single epitope on the CYP17A1 protein and are produced from a single B-cell clone, resulting in high specificity and consistency between production lots . In contrast, polyclonal antibodies like the 14447-1-AP bind to multiple epitopes on the CYP17A1 protein, potentially offering higher sensitivity but with potential for greater batch-to-batch variation .

For experiments requiring precise epitope targeting or applications where cross-reactivity is a concern, monoclonal antibodies typically provide superior specificity. Polyclonal antibodies may offer advantages in applications where the target protein is present at low concentrations, as their ability to bind multiple epitopes can enhance signal strength.

What are the validated applications for CYP17A1 antibodies?

CYP17A1 antibodies have been validated across multiple experimental techniques:

Multiple peer-reviewed publications have verified these applications, with 24 publications supporting Western blot, 15 supporting IHC, and 14 supporting IF applications for certain antibodies .

How do CYP17A1 mutations affect steroidogenic pathways and disease development?

Mutations in the CYP17A1 gene can lead to adrenal hyperplasia type V (AH-V), characterized by hypokalemia and hypertension . The pathophysiology is particularly striking in males, who may develop female external genitalia and fail to undergo normal sexual differentiation and pubertal development . This occurs because CYP17A1 mutations disrupt the 17α-hydroxylase and/or 17,20-lyase activities required for androgen production.

Recent research has expanded beyond traditional views of CYP17A1's role in adrenal disorders to investigate its involvement in other conditions. Studies using CYP17A1-deficient XY mice have demonstrated susceptibility to atherosclerosis , suggesting broader implications for cardiovascular health beyond steroid metabolism disorders.

The specific mutation location within the CYP17A1 gene determines whether both enzymatic activities are affected or just the 17,20-lyase activity, leading to varying clinical presentations. Researchers investigating CYP17A1 mutations should consider using both functional enzyme assays and protein detection methods to fully characterize the impact of specific mutations.

What role does cytochrome b5 play in regulating CYP17A1 activity?

The interaction between CYP17A1 and cytochrome b5 (cyt b5) is crucial for the selective promotion of 17,20-lyase activity . Current research suggests that cyt b5 binding alters the CYP17A1 conformation to enhance its lyase function rather than its hydroxylase activity . This conformational change does not appear to alter the binding kinetics of substrates such as 17OH-Preg or 17OH-Prog, as measured by apparent Kd and binding kinetics .

This regulatory mechanism presents a potential target for selective inhibition of androgen synthesis. The ternary complex of CYP17A1-cyt b5-POR (P450 oxidoreductase) represents a sophisticated control point in steroidogenesis, although experimental structural data regarding this complex remains limited . Researchers studying CYP17A1 regulation should consider the influence of cyt b5 when designing experiments to investigate the 17,20-lyase activity specifically.

What are the current approaches to CYP17A1 inhibition in research and therapeutic development?

CYP17A1 inhibition represents an important research area, particularly for conditions where androgen production needs to be suppressed. Non-steroidal CYP17A1 inhibitors have been developed as alternatives to steroidal inhibitors like abiraterone, with compounds such as orteronel (TAK-700) and seviteronel (VT-464) reaching clinical trials .

Recent developments in CYP17A1 inhibitors focus on achieving selectivity in three key areas:

• Selectivity against other CYPs: Compounds must avoid inhibiting drug-metabolizing CYPs such as CYP3A4 .

• Selectivity towards lyase inhibition: Ideally, inhibitors would selectively block the 17,20-lyase activity while preserving 17α-hydroxylase activity to minimize side effects .

• Structure-activity relationships: Naphthalene derivatives and compounds containing specific heme-binding heterocycles have shown promising selective inhibition profiles .

For researchers investigating CYP17A1 inhibition, it is essential to evaluate both enzymatic activities separately and assess cross-reactivity with other CYP enzymes, particularly those involved in drug metabolism.

What are the optimal conditions for Western blot detection of CYP17A1?

Successful Western blot detection of CYP17A1 requires careful optimization of several parameters:

Sample Preparation and Electrophoresis:

• Load 30 μg of sample under reducing conditions

• Use 5-20% SDS-PAGE gel

• Run at 70V (stacking gel) followed by 90V (resolving gel) for 2-3 hours

Transfer and Detection:

• Transfer to nitrocellulose membrane at 150 mA for 50-90 minutes

• Block with 5% non-fat milk/TBS for 1.5 hours at room temperature

• Incubate with primary antibody (e.g., 0.5 μg/mL for A00615-1) overnight at 4°C

• Wash with TBS-0.1% Tween (3 times, 5 minutes each)

• Probe with HRP-conjugated secondary antibody (1:5000 dilution) for 1.5 hours at room temperature

• Develop using enhanced chemiluminescent detection

CYP17A1 typically appears as a specific band at approximately 57 kDa, though observed molecular weights between 48-51 kDa have been reported . Validated positive controls include SH-SY5Y, HepG2, HeLa, and Jurkat whole cell lysates, as well as rat testis tissue lysates .

How should researchers optimize immunohistochemistry protocols for CYP17A1?

For effective CYP17A1 detection in tissue sections, the following protocol has been validated:

Tissue Processing and Antigen Retrieval:

• Use paraffin-embedded tissue sections (human testis tissue shows reliable expression)

• Perform heat-mediated antigen retrieval in citrate buffer (pH 6, epitope retrieval solution) for 20 minutes

• Alternative antigen retrieval with TE buffer pH 9.0 has also shown effectiveness

Staining Protocol:

• Block tissue section with 10% goat serum

• Incubate with primary antibody (1 μg/ml) overnight at 4°C

• Use biotinylated secondary antibody (incubation for 30 minutes at 37°C)

• Develop using Strepavidin-Biotin-Complex with DAB as the chromogen

Researchers should include appropriate positive controls (adrenal gland, testis) and negative controls (antibody omission, non-expressing tissue) to validate staining specificity.

What protocol is recommended for flow cytometric analysis of CYP17A1?

For intracellular detection of CYP17A1 by flow cytometry, researchers should follow these validated steps:

• Fix cells with 4% paraformaldehyde

• Permeabilize cells with appropriate permeabilization buffer

• Block with 10% normal goat serum

• Incubate with CYP17A1 antibody (1 μg per 10^6 cells) for 30 minutes at 20°C

• Use fluorophore-conjugated secondary antibody (e.g., DyLight®488 conjugated anti-rabbit IgG, 5-10 μg per 10^6 cells) for 30 minutes at 20°C

Essential controls include:

• Isotype control antibody (e.g., rabbit IgG at 1 μg per 10^6 cells)

• Unlabelled sample without primary and secondary antibody incubation

U87 and HepG2 cell lines have been validated for CYP17A1 detection by flow cytometry and can serve as positive controls .

How should researchers interpret multiple bands in CYP17A1 Western blots?

Multiple bands in CYP17A1 Western blots may have several legitimate explanations:

• Isoforms and splice variants: The human CYP17A1 gene can produce different transcript variants, potentially resulting in protein isoforms of varying molecular weights.

• Post-translational modifications: Phosphorylation, glycosylation, or other modifications can alter protein migration, with the calculated molecular weight being 57 kDa and observed weights often between 48-51 kDa .

• Proteolytic processing: Sample handling can lead to protein degradation, producing fragments that may be detected by the antibody.

• Non-specific binding: Some antibodies may cross-react with structurally similar proteins, particularly other CYP family members.

To distinguish between these possibilities, researchers should:

• Compare results using multiple antibodies targeting different epitopes

• Include appropriate positive control samples (e.g., HepG2, SH-SY5Y, or testis tissue)

• Consider preabsorption tests with purified antigen

• Verify results using alternative techniques like mass spectrometry

What factors influence the reproducibility of CYP17A1 antibody results?

Several critical factors affect the reproducibility of experiments using CYP17A1 antibodies:

Researchers should document all experimental conditions thoroughly and maintain consistent protocols across experiments to maximize reproducibility.

How can researchers address weak or absent signals in CYP17A1 detection?

When facing weak or absent signals in CYP17A1 detection, researchers should consider these methodological adjustments:

For Western Blotting:

• Increase protein loading (up to 50 μg per lane)

• Optimize primary antibody concentration (try 1:2000 to 1:12000 range for 14447-1-AP)

• Extend primary antibody incubation time (overnight at 4°C)

• Use enhanced chemiluminescent detection systems with higher sensitivity

• Ensure sample contains CYP17A1-expressing cells/tissues (HepG2, Jurkat, rat testis as positive controls)

For Immunohistochemistry:

• Test alternative antigen retrieval methods (compare TE buffer pH 9.0 with citrate buffer pH 6.0)

• Increase antibody concentration (try 1:100 instead of 1:400)

• Extend primary antibody incubation time (overnight at 4°C)

• Use signal amplification systems (e.g., Strepavidin-Biotin-Complex)

• Verify tissue expression (human prostate cancer and testis tissues show reliable expression)

For Flow Cytometry:

• Optimize fixation and permeabilization conditions

• Increase antibody concentration (0.40 μg per 10^6 cells is recommended)

• Test alternative fluorophores with higher brightness

• Verify expression in selected cell line (U87 cells show reliable expression)

What strategies can minimize non-specific binding with CYP17A1 antibodies?

Non-specific binding can significantly impact result interpretation. Researchers can implement these strategies to improve specificity:

• Optimize blocking conditions: Extend blocking time to 2 hours and test alternative blocking agents (BSA, serum, commercial blockers)

• Increase washing stringency: Add additional wash steps and consider higher concentrations of detergent (up to 0.3% Tween-20) in wash buffers

• Use monoclonal antibodies: Consider switching to monoclonal antibodies like D-12 for applications where cross-reactivity is problematic

• Pre-absorb antibodies: Incubate primary antibody with non-relevant tissue lysate to reduce non-specific binding

• Titrate antibody concentration: Determine the minimum concentration that provides specific signal without background (e.g., 1:2000-1:12000 for WB)

• Include competitive peptides: Use the immunizing peptide to confirm specificity of binding

Proper antibody selection based on validated applications is also crucial - consider the extensive validation data available for antibodies like 14447-1-AP, A00615-1, and D-12 across various techniques and sample types .