CYP21-2 Antibody

What is CYP21A2 and what is its biological significance?

CYP21A2 is a cytochrome P450 monooxygenase that plays a critical role in adrenal steroidogenesis. It catalyzes the hydroxylation at C-21 of progesterone and 17alpha-hydroxyprogesterone to form 11-deoxycorticosterone and 11-deoxycortisol, respectively. These are intermediate metabolites in the biosynthetic pathway of mineralocorticoids and glucocorticoids . Mechanistically, CYP21A2 utilizes molecular oxygen, inserting one oxygen atom into its substrate while reducing the second into a water molecule. This process requires two electrons provided by NADPH via cytochrome P450 reductase . Mutations in the CYP21A2 gene cause the most common form of congenital adrenal hyperplasia (CAH), an autosomal recessive disorder affecting steroid hormone synthesis .

What types of CYP21A2 antibodies are available for research?

CYP21A2 antibodies used in research are primarily polyclonal or monoclonal antibodies raised against specific epitopes of the human CYP21A2 protein. For instance, rabbit polyclonal antibodies targeting recombinant fragments within the human CYP21A2 protein (typically amino acids 100-400) are commonly used . These antibodies are validated for various applications including Western blotting (WB) and immunohistochemistry on paraffin-embedded tissues (IHC-P), and can react with human and rat samples . When selecting an antibody, researchers should consider the species reactivity, the specific epitope recognized, and validated applications to ensure compatibility with their experimental design.

What are the common applications for CYP21A2 antibodies in research?

CYP21A2 antibodies are primarily used in the following research applications:

• Western blotting - For detection and quantification of CYP21A2 protein expression levels

• Immunohistochemistry - For localization of CYP21A2 in tissue sections, particularly adrenal tissues

• Immunofluorescence - For cellular localization studies

• Immunoprecipitation - For protein-protein interaction studies involving CYP21A2

• ELISA - For quantitative measurement of CYP21A2 in biological samples

These antibodies are particularly valuable in CAH research, steroid hormone metabolism studies, and investigations of adrenal gland physiology and pathology . When using CYP21A2 antibodies for protein detection, appropriate positive and negative controls should be included to validate specificity.

How can CYP21A2 antibodies be used to characterize humanized mouse models?

CYP21A2 antibodies serve as essential tools for validating humanized CYP21A2 mouse models. In these models, the mouse Cyp21a1 gene is replaced with the human CYP21A2 gene using techniques such as CRISPR/Cas9-mediated gene targeting . To properly characterize such models, researchers should:

• Use CYP21A2 antibodies in immunohistochemistry to confirm expression patterns in adrenal glands

• Perform Western blot analysis to verify protein expression levels and compare with wild-type controls

• Conduct immunofluorescence to examine subcellular localization

• Apply co-immunoprecipitation to investigate protein-protein interactions

When examining homozygous CYP21A2 mice, antibody detection should confirm the absence of mouse Cyp21a1 and the presence of human CYP21A2 in adrenal tissues. These models show analogous expression patterns of human 21-hydroxylase compared to murine 21-hydroxylase in wild-type animals, making antibody specificity crucial for accurate characterization .

How can researchers use CYP21A2 antibodies to study mutations linked to congenital adrenal hyperplasia?

CYP21A2 antibodies are valuable tools for investigating how mutations affect protein expression, stability, and localization in CAH research. Researchers can:

• Express wild-type and mutant CYP21A2 in cell models (e.g., COS-1 cells)

• Use antibodies to compare expression levels between wild-type and mutant proteins

• Assess protein stability through pulse-chase experiments with immunoprecipitation

• Determine subcellular localization changes using immunofluorescence

For functional studies, antibodies can complement enzyme activity assays. For example, reduced enzyme activities between 43% and 97% were observed for mutations p.Arg16Cys, p.Ser101Asn, p.Ser202Gly, p.Pro267Leu, and p.Thr450Met, similar to known nonclassic mutations p.Pro453Ser and p.Pro482Ser . Severe mutations like p.Ser113Phe, p.Gln389_Ala391del, and p.Thr450Pro dramatically reduce enzyme function below 4% . Antibodies can help correlate these functional changes with protein expression levels, providing insight into whether loss of function is due to protein instability or catalytic defects.

What methodologies combine CYP21A2 antibodies with genetic analysis for comprehensive CAH research?

A comprehensive approach to CAH research integrates genetic analysis with protein-level studies using CYP21A2 antibodies:

• Perform genetic analysis using locus-specific PCR, Sanger sequencing, and MLPA to identify mutations

• Use CYP21A2 antibodies to assess protein expression in patient-derived samples

• Develop in vitro expression systems for wild-type and mutant proteins

• Apply antibodies in Western blot, immunoprecipitation, and immunofluorescence to characterize mutant proteins

• Correlate genotype, protein expression, and clinical phenotype

The combination of molecular techniques (PCR, sequencing) with immunological methods provides a comprehensive understanding of how genetic variations affect protein function and ultimately clinical presentation . For example, researchers investigating two rare variant alleles in a 22-year-old female with non-salt-wasting CAH used allele-specific PCR and sequencing complemented with protein expression analysis to understand the molecular basis of the phenotype .

What are the optimal conditions for Western blotting with CYP21A2 antibodies?

For successful Western blotting with CYP21A2 antibodies, follow these methodological guidelines:

• Sample preparation:

  • Extract proteins from tissues (adrenal glands preferred) or transfected cells

  • Include protease inhibitors to prevent degradation

  • Use 20-50 μg of total protein per lane

• Gel electrophoresis and transfer:

  • Use 10-12% SDS-PAGE gels

  • Transfer to PVDF membranes (preferred over nitrocellulose for hydrophobic proteins)

  • Transfer at 100V for 1 hour or 30V overnight at 4°C

• Antibody incubation:

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

  • Dilute primary antibody 1:1000 to 1:2000 in blocking buffer

  • Incubate overnight at 4°C with gentle rocking

  • Wash 3-5 times with TBST

  • Use appropriate HRP-conjugated secondary antibody (1:5000-1:10000)

  • Incubate for 1 hour at room temperature

• Detection:

  • Use enhanced chemiluminescence (ECL) substrate

  • Expect a band at approximately 55-56 kDa for CYP21A2

Include appropriate positive controls (adrenal tissue or CYP21A2-expressing cell lines) and negative controls (tissues not expressing CYP21A2 or knockdown samples) .

What protocols yield optimal results for immunohistochemistry with CYP21A2 antibodies?

For effective immunohistochemistry with CYP21A2 antibodies, follow this optimized protocol:

• Tissue preparation:

  • Fix tissues in 10% neutral buffered formalin for 24-48 hours

  • Process and embed in paraffin

  • Section at 4-5 μm thickness

• Antigen retrieval:

  • Deparaffinize and rehydrate sections

  • Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Heat in pressure cooker or microwave for 10-20 minutes

  • Cool to room temperature

• Immunostaining:

  • Block endogenous peroxidase with 3% H₂O₂ for 10 minutes

  • Block non-specific binding with 5% normal serum from secondary antibody species

  • Apply CYP21A2 primary antibody at 1:100-1:200 dilution

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

  • Wash thoroughly with PBS

  • Apply appropriate secondary antibody (30-60 minutes)

  • Develop with DAB and counterstain with hematoxylin

• Controls:

  • Include positive control (normal adrenal tissue)

  • Include negative control (omit primary antibody)

  • Consider using tissues from CYP21A2 knockout models as specificity controls

CYP21A2 antibodies typically show strong cytoplasmic staining in the zona fasciculata and zona reticularis of the adrenal cortex, corresponding to the subcellular localization of this enzyme in the endoplasmic reticulum .

How can researchers validate the specificity of CYP21A2 antibodies?

Validating CYP21A2 antibody specificity is crucial for reliable research results. Apply these methodological approaches:

• Genetic validation:

  • Use tissues/cells from CYP21A2 knockout models as negative controls

  • Compare staining patterns between wild-type and knockout samples

  • Use humanized CYP21A2 mouse models with species-specific antibodies

• Expression validation:

  • Use siRNA or shRNA knockdown of CYP21A2 to reduce expression

  • Compare antibody signals before and after knockdown

  • Overexpress CYP21A2 in non-expressing cell lines and confirm signal increase

• Peptide competition:

  • Pre-incubate antibody with the immunizing peptide

  • Apply this mixture in parallel with regular antibody

  • Specific signals should be abolished or reduced

• Cross-reactivity assessment:

  • Test the antibody against related cytochrome P450 enzymes

  • Express CYP21A2 and related proteins in the same system

  • Confirm selective detection of CYP21A2

For comprehensive validation, combine multiple approaches and document all validation steps in scientific publications. Proper validation ensures reliable interpretation of results, particularly when studying closely related proteins or complex biological samples .

How can researchers resolve common issues with CYP21A2 antibody detection in Western blotting?

When facing challenges with CYP21A2 detection in Western blotting, consider these methodological solutions:

When working with adrenal tissue samples, consider using a microsomal preparation rather than whole cell lysates to enrich for CYP21A2, as it is associated with the endoplasmic reticulum .

What are the key considerations when using CYP21A2 antibodies in co-immunoprecipitation studies?

For successful co-immunoprecipitation (co-IP) experiments with CYP21A2 antibodies, researchers should follow these methodological guidelines:

• Lysis conditions:

  • Use mild detergents (0.5-1% NP-40, Triton X-100, or digitonin)

  • Include protease inhibitors and phosphatase inhibitors

  • Maintain physiological pH (7.4-7.6)

  • Keep samples cold (4°C) throughout the procedure

• Pre-clearing step:

  • Incubate lysate with protein A/G beads without antibody

  • Rotate for 1 hour at 4°C

  • Remove beads by centrifugation to reduce non-specific binding

• Antibody selection and binding:

  • Choose antibodies validated for immunoprecipitation

  • Use 2-5 μg antibody per 500 μg protein lysate

  • Incubate antibody with lysate for 2-4 hours or overnight at 4°C

  • Add pre-washed protein A/G beads and rotate for 1-2 hours

• Washing and elution:

  • Wash beads 4-5 times with lysis buffer containing reduced detergent

  • Elute proteins with SDS sample buffer at 95°C for 5 minutes

• Controls:

  • Include isotype control antibody IP

  • Perform reverse co-IP if possible

  • Save input, unbound, and wash fractions for troubleshooting

CYP21A2 interacts with other steroidogenic enzymes and electron transfer proteins in the endoplasmic reticulum, making co-IP studies valuable for investigating these protein-protein interactions . When studying CYP21A2 mutations, co-IP can reveal altered interactions that may contribute to disease mechanisms.

How can CYP21A2 antibodies be used to compare wild-type and mutant protein expression in transfected cell models?

For comparative analysis of wild-type and mutant CYP21A2 protein expression, follow this methodological framework:

• Expression system preparation:

  • Select appropriate cell line (COS-1, HEK293, or H295R adrenal cells)

  • Create expression constructs for wild-type and mutant CYP21A2

  • Ensure identical promoters and tags for comparable expression

  • Transfect cells using identical conditions for all constructs

• Expression analysis:

  • Harvest cells 24-48 hours post-transfection

  • Prepare parallel samples for Western blot and enzyme activity

  • Run wild-type and mutants on the same gel for direct comparison

  • Use house-keeping proteins (GAPDH, β-actin) for normalization

  • Quantify band intensity using densitometry software

• Subcellular localization:

  • Perform immunofluorescence with CYP21A2 antibodies

  • Co-stain with organelle markers (e.g., calnexin for ER)

  • Analyze by confocal microscopy to detect mislocalization

• Protein stability assessment:

  • Treat cells with cycloheximide to inhibit protein synthesis

  • Harvest at time points (0, 2, 4, 8, 24 hours)

  • Compare degradation rates between wild-type and mutants

This approach has been successfully employed to study mutations such as p.Arg16Cys, p.Ser101Asn, p.Ser202Gly, p.Pro267Leu, and p.Thr450Met, which showed residual enzyme activities between 43% and 97%, and severe mutations like p.Ser113Phe, p.Gln389_Ala391del, and p.Thr450Pro that reduce function below 4% . Correlated with clinical data, such studies provide invaluable insights into genotype-phenotype relationships in CAH.

How can researchers effectively combine genetic testing and antibody-based protein analysis in CAH research?

An integrated approach combining genetic and protein analysis provides comprehensive insights into CAH pathophysiology:

• Sequential methodology:

  • Begin with genetic screening for common mutations using locus-specific PCR

  • Perform MLPA to detect large gene deletions/conversions

  • Use Sanger sequencing to identify point mutations

  • Apply antibody-based techniques to analyze protein consequences

• Sample coordination:

  • Collect paired samples for DNA and protein analysis

  • Establish lymphoblastoid cell lines from patients for extended studies

  • Consider skin fibroblasts for protein expression in patient-derived cells

• Data integration:

  • Correlate mutation location with protein structure and function

  • Use antibodies to confirm predicted protein effects

  • Compare protein expression levels with enzyme activity measurements

  • Relate molecular findings to clinical severity

This integrated approach has been successfully applied in studies where mutations such as micro-conversion mutation IVS2-13A/C>G (found in 70.5% of alleles), large gene deletions and conversions (22.7%), p.R357W (4.5%), and E6 Cluster (2.3%) were identified and correlated with clinical phenotypes in salt-wasting 21-OHD patients . The combined genetic-protein analysis provides a more complete understanding of disease mechanisms than either approach alone.

What novel approaches combine CYP21A2 antibodies with structural biology techniques?

Advanced research integrating antibody-based methods with structural biology offers new insights into CYP21A2 function:

• Epitope mapping and structural correlation:

  • Use deletion mutants or peptide arrays with CYP21A2 antibodies

  • Map antibody binding sites onto the crystallized human CYP21A2 structure

  • Correlate antigenic regions with functional domains

• Conformational antibodies:

  • Develop antibodies that recognize specific conformational states

  • Use these to study protein dynamics during catalytic cycle

  • Apply in studies of how mutations affect protein folding

• Proximity labeling:

  • Combine antibodies with proximity labeling techniques (BioID, APEX)

  • Identify proteins in the CYP21A2 microenvironment

  • Map the spatial organization of steroidogenic enzyme complexes

• Cryo-EM applications:

  • Use antibodies as fiducial markers for cryo-EM studies

  • Study CYP21A2 in complex with electron transfer partners

  • Visualize structural changes in disease-causing mutations

These approaches leverage the recently crystallized human CYP21A2 structure to provide deeper insights into how mutations affect enzyme function . For example, functional analyses complemented with in silico prediction of mutation pathogenicity based on this structure have helped understand how specific residues contribute to enzyme activity and substrate binding.