CYP21-1 Antibody

What is CYP21A2 and why is it important in research?

CYP21A2 (cytochrome P450, family 21, subfamily A, polypeptide 2) is a crucial enzyme involved in steroid hormone biosynthesis, specifically catalyzing the 21-hydroxylation of steroids in the adrenal cortex. This enzyme is essential for the production of cortisol and aldosterone. Mutations in the CYP21A2 gene are responsible for more than 90% of congenital adrenal hyperplasia (CAH) cases, making it a significant target for endocrinological research . The study of CYP21A2 and antibodies against it allows researchers to investigate steroid metabolism, adrenal function, and pathophysiological mechanisms underlying CAH.

What is the difference between CYP21 and CYP21P genes?

The CYP21 gene (now officially designated CYP21A2) is the functional gene that encodes the active 21-hydroxylase enzyme. In contrast, CYP21P is a highly homologous pseudogene that contains various deleterious mutations, including frameshift mutations and premature stop codons, rendering it non-functional . While CYP21P shares approximately 98% sequence identity with CYP21A2 in exons, the pseudogene cannot produce functional enzyme due to these inactivating mutations. Intergenic recombination between CYP21A2 and CYP21P is responsible for many cases of 21-hydroxylase deficiency, creating chimeric genes that contribute to CAH pathogenesis .

What are the molecular characteristics of CYP21A2 protein?

The CYP21A2 protein is a microsomal cytochrome P450 enzyme with a calculated molecular weight of 56 kDa, though it typically appears at 53-56 kDa in experimental conditions . The protein's native form is associated with the endoplasmic reticulum membrane of adrenocortical cells. As a member of the cytochrome P450 superfamily, it contains a heme prosthetic group essential for its hydroxylation activity. The gene encoding CYP21A2 is located on chromosome 6p21.3 as part of the HLA complex, with GenBank accession number NM_000500 and UNIPROT ID P08686 .

What are the optimal experimental conditions for Western blot detection of CYP21A2?

For Western blot applications using CYP21A2 antibody (67421-1-Ig), researchers should implement the following protocol:

• Sample preparation: Extract proteins from target tissues (adrenal gland recommended) or cell lines (HepG2 or PC-12 cells have demonstrated positive expression)

• Protein loading: 20-40 μg of total protein per lane is typically sufficient

• Antibody dilution: Use at 1:2000-1:10000 dilution in blocking buffer

• Positive controls: Include HepG2 cells, PC-12 cells, or pig adrenal gland tissue as positive controls

• Expected molecular weight: Look for bands at 53-56 kDa

• Optimization: Sample-dependent optimization may be necessary to minimize background and maximize specific signal

When troubleshooting, consider longer exposure times for weaker signals and include appropriate negative controls to confirm specificity.

How can researchers effectively use CYP21A2 antibodies for immunofluorescence studies?

For optimal immunofluorescence/immunocytochemistry applications with CYP21A2 antibody:

• Sample preparation:

  • For cultured cells: Fix with 4% paraformaldehyde for 15 minutes at room temperature

  • For tissue sections: Use freshly prepared or properly preserved specimens

• Antibody application:

  • Use at 1:400-1:1600 dilution for IF/ICC applications

  • Incubate with primary antibody (67421-1-Ig) overnight at 4°C

  • PC-12 cells serve as reliable positive controls

• Detection system:

  • Use appropriate fluorescently-labeled secondary antibodies

  • Include DAPI for nuclear counterstaining

  • Examine subcellular localization (typically endoplasmic reticulum pattern)

• Controls and validation:

  • Include negative controls (secondary antibody only)

  • Consider dual staining with ER markers to confirm subcellular localization

The optimal protocol should be validated and potentially modified based on specific experimental conditions and cell/tissue types.

What methods are available for detecting chimeric CYP21P/CYP21 genes in research samples?

Detection of chimeric CYP21P/CYP21 genes requires specialized molecular approaches due to their complex nature:

• PCR-based detection:

  • Allele-specific PCR using primers specific to CYP21P and CYP21 regions (e.g., BF1, AF1, and 21BR primers in combination)

  • Expected PCR product size: ~3.5 kb covering sequences from -313 to 3170

• Restriction fragment length polymorphism (RFLP) analysis:

  • Digest PCR products with restriction enzymes AflII, AlwI, and AseI

  • Normal CYP21 gene shows fragments of 1705, 898, 796, and 85 bp

  • Chimeric genes show a distinctive pattern (1705, 899, 696, 115, and 85 bp)

• Amplification-created restriction site (ACRS) method:

  • Use specific ACRS primer pairs for detecting key mutations (e.g., P30L, nt 655, G110delGA-Y112del)

  • Perform restriction digestion followed by gel electrophoresis analysis

• Southern blot analysis:

  • PCR products amplified with allele-specific primers covering TNXB to the 5' end of CYP21 gene

  • Combined with digestion using AseI and NdeI for chimera identification

These molecular techniques offer complementary approaches for comprehensive analysis of CYP21 genetic variants in research settings.

How can CYP21A2 antibodies be used to investigate congenital adrenal hyperplasia mechanisms?

CYP21A2 antibodies serve as powerful tools for investigating CAH pathophysiology through multiple research approaches:

• Protein expression analysis:

  • Quantify CYP21A2 levels in patient-derived samples compared to controls

  • Correlate protein expression with clinical severity and genotype

  • Examine expression in various tissues beyond adrenal (e.g., gonads)

• Functional studies:

  • Immunoprecipitate active enzyme for in vitro activity assays

  • Correlate protein levels with enzymatic activity in different CAH variants

  • Study protein-protein interactions involving CYP21A2 in steroidogenic complexes

• Cellular localization:

  • Examine subcellular trafficking of mutant CYP21A2 proteins

  • Investigate potential misfolding and ER retention of mutant proteins

  • Compare wild-type versus mutant protein localization patterns

• Therapeutic development:

  • Monitor protein expression in gene therapy approaches

  • In AAV-mediated gene transfer studies, assess dose-dependent expression of human CYP21A2 protein in adrenal tissue

  • Validate vector-mediated expression at the protein level alongside mRNA and vector genome copy number analyses

This multifaceted approach provides comprehensive insights into CAH pathogenesis and potential therapeutic interventions.

What are the key considerations when using CYP21A2 antibodies in gene therapy validation studies?

When employing CYP21A2 antibodies to validate gene therapy approaches for 21-hydroxylase deficiency:

• Expression level assessment:

  • Quantify CYP21A2 protein in treated versus untreated tissues

  • Determine dose-response relationships between vector dose and protein expression

  • Compare protein levels to endogenous expression in wild-type models (percentage of normal)

• Methodology optimization:

  • Select appropriate antibody dilutions based on expected expression levels

  • Consider tissue-specific protocols for protein extraction from adrenal samples

  • Implement quantitative Western blotting with appropriate standards

• Correlative analyses:

  • Correlate protein expression with:

    • Vector genome copies per μg genomic DNA

    • CYP21A2 mRNA levels

    • Physiological parameters (e.g., steroid hormone levels)

    • Clinical improvements (e.g., weight gain in animal models)

• Species considerations:

  • Ensure antibody cross-reactivity with the species under investigation

  • Consider species-specific controls when evaluating human CYP21A2 expression in animal models

  • Validate antibody performance in the specific experimental context

These considerations ensure robust protein-level validation of gene therapy approaches targeting 21-hydroxylase deficiency.

How can researchers differentiate between normal CYP21A2 and chimeric CYP21P/CYP21 proteins?

Distinguishing between normal and chimeric proteins poses significant analytical challenges requiring sophisticated approaches:

• Epitope mapping:

  • Select antibodies recognizing epitopes in regions likely to differ between normal and chimeric proteins

  • Consider antibodies targeting N-terminal regions where chimeric junctions often occur

  • Use multiple antibodies recognizing different domains for comparative analysis

• Molecular weight analysis:

  • Chimeric genes with frameshift mutations (e.g., G110delGA-Y112del) produce truncated proteins

  • High-resolution SDS-PAGE can resolve potential size differences

  • Consider using gradient gels for optimal separation of closely migrating species

• Functional assays:

  • Combine immunological detection with activity assays

  • Chimeric proteins typically show reduced or absent enzymatic activity

  • Correlate protein detection with functional readouts

• Mass spectrometry:

  • Employ immunoprecipitation followed by mass spectrometry

  • Identify peptide sequences unique to normal versus chimeric proteins

  • Analyze post-translational modification patterns that may differ

These advanced approaches enable researchers to distinguish normal CYP21A2 from chimeric variants at the protein level, complementing genetic analyses.

How can researchers address cross-reactivity issues when studying CYP21A2 in complex samples?

When working with CYP21A2 antibodies in complex biological samples:

• Antibody validation steps:

  • Test antibody specificity using recombinant CYP21A2 protein

  • Include known positive controls (HepG2 cells, PC-12 cells, pig adrenal tissue)

  • Perform peptide competition assays to confirm specific binding

• Sample preparation optimization:

  • Enrich for microsomes/endoplasmic reticulum fraction when possible

  • Consider immunoprecipitation prior to immunoblotting for complex samples

  • Optimize extraction buffers to maintain protein integrity while minimizing interference

• Detection strategies:

  • Implement more stringent washing protocols to reduce non-specific binding

  • Adjust blocking conditions (duration, composition) to minimize background

  • Consider alternative detection systems with higher specificity

• Controls and comparisons:

  • Include tissues known to lack CYP21A2 expression as negative controls

  • Compare results from multiple antibody clones when available

  • Validate findings using complementary methodologies (e.g., mass spectrometry)

These strategies help ensure specific detection of CYP21A2 while minimizing potential cross-reactivity with other cytochrome P450 family members.

What are the best practices for sample preparation to maximize CYP21A2 antibody performance?

Optimal sample preparation significantly impacts antibody performance in CYP21A2 detection:

• Tissue/cell lysis considerations:

  • Use buffers containing mild detergents (e.g., 0.5-1% Triton X-100) to solubilize membrane-associated CYP21A2

  • Include protease inhibitors to prevent degradation

  • Consider gentle homogenization methods to preserve protein structure

• Storage recommendations:

  • Store antibody at -20°C with 50% glycerol and 0.02% sodium azide

  • Aliquoting is generally unnecessary for -20°C storage of small volumes (e.g., 20μl)

  • For long-term sample storage, maintain at -80°C with cryoprotectants

• Application-specific considerations:

  • For Western blot: Denature samples completely but avoid excessive heating

  • For immunofluorescence: Optimize fixation conditions (PFA concentration, duration)

  • For immunoprecipitation: Consider native conditions to maintain enzymatic activity

• Quality control:

  • Monitor protein integrity using total protein stains

  • Include housekeeping proteins as loading controls

  • Consider tissue-specific markers to confirm sample quality

Following these best practices ensures optimal extraction, preservation, and detection of CYP21A2 protein across different experimental applications.

How might single-cell approaches advance understanding of CYP21A2 function and pathology?

Emerging single-cell technologies offer promising avenues for investigating CYP21A2 biology:

• Single-cell protein analysis:

  • Employ CyTOF or microfluidic antibody-based techniques to quantify CYP21A2 at the single-cell level

  • Identify cell-to-cell variability in expression within adrenal tissues

  • Correlate with steroidogenic enzyme co-expression patterns

• Spatial transcriptomics integration:

  • Combine CYP21A2 antibody-based imaging with spatial transcriptomics

  • Map protein expression to transcriptional profiles within tissue architecture

  • Identify microenvironmental factors influencing expression

• Patient-derived models:

  • Analyze CYP21A2 expression in patient-derived organoids or iPSC models

  • Investigate cell-type specific responses to treatments at the single-cell level

  • Model developmental trajectories of CYP21A2-expressing adrenocortical cells

• Methodological adaptations:

  • Optimize antibody protocols for single-cell Western blotting techniques

  • Develop multiplexed antibody panels including CYP21A2 and related proteins

  • Implement computational approaches for integrating single-cell protein and transcriptomic data

These innovative approaches represent the frontier of CYP21A2 research, potentially revealing new insights into adrenal biology and pathology.

What role might CYP21A2 antibodies play in validating emerging gene therapy approaches for CAH?

CYP21A2 antibodies will be instrumental in advancing gene therapy approaches for 21-hydroxylase deficiency:

• Preclinical validation:

  • Quantify CYP21A2 protein expression following AAV-mediated gene transfer

  • Establish dose-response relationships between vector dose and protein expression

  • Current data indicate detection of human CYP21A2 mRNA and protein in adrenal glands following AAV5-based treatment

• Translational research applications:

  • Monitor protein expression in ongoing clinical trials

  • Correlate protein levels with clinical endpoints and biomarkers

  • First-in-human studies using AAV-mediated gene transfer for CAH are underway with planned dose escalation (1.5 × 10¹³, 3.0 × 10¹³, and 6.0 × 10¹³ vg/kg)

• Long-term expression monitoring:

  • Assess stability of transgene protein expression over time

  • Detect potential adaptive immune responses to expressed protein

  • Evaluate integration with endogenous steroidogenic pathways

• Comparative analysis:

  • Compare protein expression between different gene delivery vectors

  • Assess promoter effects on tissue-specific expression patterns

  • Evaluate expression in different genetic backgrounds

These applications highlight the critical role of CYP21A2 antibodies in advancing gene therapy from preclinical models to clinical implementation.

What dilutions and experimental conditions are recommended for different applications of CYP21A2 antibody?

The following table summarizes recommended experimental conditions for CYP21A2 antibody (67421-1-Ig) applications:

Storage Buffer: PBS with 0.02% sodium azide and 50% glycerol, pH 7.3

Storage Conditions: Store at -20°C; stable for one year after shipment; aliquoting unnecessary for -20°C storage of small volumes

Protein Molecular Weight: Expected at 53-56 kDa (calculated: 56 kDa)

These specifications provide standardized parameters for antibody use while acknowledging the need for optimization based on specific experimental systems.

What molecular characteristics distinguish chimeric CYP21P/CYP21 genes in research samples?

The following features can be used to identify and characterize chimeric CYP21P/CYP21 genes:

• Structural characteristics:

  • Result from unequal crossover between CYP21P and CYP21 genes

  • 5' end corresponds to CYP21P pseudogene and 3' end to functional CYP21

  • Typically arise from 26-kb or 32-kb deletions in C4-CYP21 repeat module

• Junction points:

  • Can occur at multiple sites, including:

    • Chi-like sequence GCTGGGC in the third intron

    • Minisatellite consensus TGGCAGGAGG in exon 5

  • At least three distinct chimeras identified in ethnic Chinese CAH patients

• Diagnostic restriction patterns:

  • CYP21P gene: four fragments of 2599, 688, 115, and 85 bp (AflII/AlwI/AseI digestion)

  • Normal CYP21 gene: 1705, 898, 796, and 85 bp fragments (AflII/AlwI digestion)

  • Chimeric genes: 1705, 899, 696, 115, and 85 bp fragments

• Functional consequences:

  • Typically nonfunctional due to deleterious mutations like G110delGA-Y112del

  • Results in frameshift mutation producing truncated protein

  • Complete loss of 21-hydroxylase enzymatic activity