CYP51G1 Antibody

What is CYP51/CYP51A1 and what role does it play in cellular metabolism?

CYP51A1 (also known as Lanosterol 14-alpha demethylase) is a critical enzyme belonging to the evolutionarily conserved cytochrome P450 family. It functions as a sterol 14alpha-demethylase that plays an essential role in the cholesterol biosynthesis pathway. Cholesterol serves as the major sterol component in mammalian membranes and acts as a precursor for bile acid and steroid hormone synthesis .

At the molecular level, CYP51A1 is a cytochrome P450 monooxygenase that catalyzes a three-step oxidative reaction: it removes the 14alpha-methyl group (C-32) of sterols such as lanosterol (lanosta-8,24-dien-3beta-ol) and 24,25-dihydrolanosterol (DHL) in the form of formate. This process converts the sterols to 4,4-dimethyl-5alpha-cholesta-8,14,24-trien-3beta-ol and 4,4-dimethyl-8,14-cholestadien-3beta-ol respectively, which are key intermediates in the cholesterol biosynthesis pathway .

Interestingly, CYP51A1 can also demethylate substrates not intrinsic to mammals, such as eburicol (24-methylene-24,25-dihydrolanosterol), though at a lower catalytic rate than its primary substrate DHL .

What applications can CYP51 antibodies be used for in laboratory research?

CYP51 antibodies have been validated for multiple research applications, with specific documented uses including:

When designing experiments, researchers should note that optimal antibody dilutions may require titration in each specific testing system to obtain optimal results . The extensive validation in published literature (with over 26 publications documenting Western blot applications alone) provides strong confidence in the reliability of these reagents for detecting CYP51 across multiple experimental platforms .

What species reactivity can be expected with commercial CYP51 antibodies?

Commercial CYP51 antibodies exhibit cross-reactivity with samples from multiple species due to the high conservation of this protein across evolutionary lines. The documented reactivity includes:

The observed molecular weight of human CYP51A1 is approximately 55 kDa, closely matching its calculated molecular weight of 57 kDa (509 amino acids) . When planning cross-species studies, it is advisable to review sequence homology between target species and the immunogen used to generate the antibody to predict potential reactivity .

What are the optimal storage conditions for maintaining CYP51 antibody activity?

Proper storage of CYP51 antibodies is crucial for maintaining their specificity and sensitivity in experimental applications. Based on manufacturer recommendations:

CYP51 antibodies should be stored at -20°C, where they typically remain stable for one year after shipment . The typical storage buffer consists of PBS with 0.02% sodium azide and 50% glycerol at pH 7.3, which helps maintain antibody integrity during freeze-thaw cycles .

How can researchers validate the specificity of CYP51 antibodies in their experimental systems?

Validating antibody specificity is critical for ensuring reliable research outcomes. For CYP51 antibodies, a comprehensive validation approach includes:

• Knockdown/Knockout Controls: Several publications have documented the use of CYP51 knockdown or knockout systems as negative controls. This approach provides the most stringent validation of antibody specificity .

• Western Blot Analysis: Verify a single band at the expected molecular weight (approximately 55 kDa for human CYP51A1). Multiple or unexpected bands may indicate cross-reactivity .

• Immunoprecipitation Validation: Confirm that the antibody can specifically pull down CYP51 from complex protein mixtures. This has been validated for certain commercial antibodies in mouse testis and heart tissues .

• Comparison with Commercial Standards: When developing new detection methods, researchers should compare their results with commercially available CYP51 protein standards. For example, studies comparing synthesized CYP51 protein with commercial preparations have shown significant differences in detection sensitivity, highlighting the importance of protein quality in antibody-based assays .

• Cross-Validation with Multiple Antibodies: Using antibodies from different manufacturers or those targeting different epitopes can provide additional confidence in experimental findings.

What controls should be included when using CYP51 antibodies for immunohistochemistry or immunofluorescence?

When performing immunohistochemistry (IHC) or immunofluorescence (IF) with CYP51 antibodies, the following controls are essential:

• Negative Controls:

  • Primary antibody omission: Apply only secondary antibody to identify non-specific binding

  • Isotype controls: Use non-specific IgG from the same species and at the same concentration

  • Tissue negative controls: Include tissues known not to express CYP51 or with very low expression levels

• Positive Controls:

  • Tissues with documented high CYP51 expression (e.g., liver tissue shows strong reactivity)

  • Cell lines with validated CYP51 expression (e.g., HeLa cells, HuH-7 cells)

• Technical Validation:

  • Antibody dilution series to determine optimal working concentration

  • Antigen retrieval optimization, particularly for formalin-fixed tissues

  • Blocking optimization to minimize background staining

• Biological Validation:

  • Comparison with mRNA expression data from the same samples

  • Correlation with functional assays of CYP51 activity

What protein-protein interactions involving CYP51 have been documented, and how can they be studied?

CYP51 functions within complex enzymatic networks in the cholesterol biosynthesis pathway. Several protein interactions have been documented:

These interactions can be studied using various methodologies:

• Co-immunoprecipitation (Co-IP): CYP51 antibodies can be used to pull down protein complexes from cell lysates. This approach has successfully identified interaction partners in cytochrome P450 family studies .

• Proximity Ligation Assay (PLA): This technique can detect protein interactions in situ within fixed cells or tissues, providing spatial information about where CYP51 interactions occur.

• Bimolecular Fluorescence Complementation (BiFC): By tagging CYP51 and potential partners with complementary fluorescent protein fragments, researchers can visualize interactions in living cells.

• Membrane Yeast Two-Hybrid Systems: Since CYP51 is a membrane-associated protein, specialized yeast two-hybrid systems designed for membrane proteins may be more appropriate than classical methods .

When designing protein interaction studies, it's essential to consider the membrane association of CYP51 and its location in the endoplasmic reticulum. Detergent selection and solubilization conditions can significantly impact the detection of genuine interaction partners versus artifacts .

How do post-translational modifications affect CYP51 detection with antibodies?

Post-translational modifications (PTMs) can significantly influence antibody recognition of CYP51. Key considerations include:

• Phosphorylation Sites: CYP51 contains multiple potential phosphorylation sites that may affect protein conformation and epitope accessibility. Researchers should be aware that phosphorylation status could affect antibody binding efficiency.

• Glycosylation: Though not extensively documented for CYP51, glycosylation of nearby residues might impact antibody binding.

• Nitric Oxide Modifications: Evidence suggests that CYP51A1 protein can be targeted for degradation when exposed to nitric oxide generated under inflammatory conditions by NOS2 or released from NO donor compounds . This degradation pathway may affect detection in samples from inflammatory conditions.

• Protein Degradation Products: When studying CYP51 in complex biological samples, researchers should be attentive to potential degradation products that might be detected as additional bands in Western blots.

To account for these effects, researchers can:

• Use phosphatase treatments to remove phosphorylation

• Compare results from antibodies targeting different epitopes

• Include appropriate controls from tissues or cells with altered PTM status

What approaches can resolve contradictory results between different detection methods for CYP51?

When facing discrepancies in CYP51 detection across different methods, consider the following reconciliation approaches:

• Antibody Comparison and Validation:

  • Different antibodies may target distinct epitopes that are differentially accessible in various applications

  • Systematically validate each antibody using knockout/knockdown controls specific to your experimental system

  • Compare results from multiple antibodies targeting different regions of CYP51

• Method-Specific Optimizations:

  • For Western blotting: Optimize protein extraction methods specifically for membrane proteins

  • For IHC/IF: Test multiple fixation and antigen retrieval protocols

  • For ELISA: Validate standard curves with recombinant proteins and assess matrix effects

• Sample Preparation Considerations:

  • CYP51 is membrane-associated and requires appropriate detergents for solubilization

  • Different tissues may require specialized extraction protocols

  • Consider the impact of sample storage conditions on epitope preservation

• Complementary Approaches:

  • Supplement antibody-based detection with mass spectrometry

  • Correlate protein detection with mRNA expression

  • Assess enzyme activity as a functional readout

A systematic investigation comparing antibody performance across multiple tissues found that synthesized CYP51 protein used as an immunogen produced significantly higher detection sensitivity than commercially available standards, highlighting how reagent quality can impact experimental outcomes .

How does CYP51 expression vary across tissues and what implications does this have for immunodetection?

CYP51 expression exhibits tissue-specific patterns that researchers should consider when designing detection protocols:

• High Expression Tissues:

  • Liver: Shows robust CYP51 expression, making it ideal as a positive control tissue

  • Testis: Demonstrates strong and consistent CYP51 expression, useful for immunoprecipitation validation

  • Heart: Also shows detectable expression suitable for immunoprecipitation studies

• Detection Sensitivity Requirements:

  • High-expressing tissues: Standard Western blot protocols are typically sufficient

  • Low-expressing tissues: May require enhanced detection methods such as chemiluminescent substrates with longer exposure times or amplification steps

• Background Considerations:

  • Tissues with high endogenous peroxidase activity (e.g., liver) may require additional blocking steps for IHC

  • Tissues with high lipid content may show increased background and require optimized washing procedures

Studies using commercial antibodies have successfully detected CYP51 in multiple human cell lines (HeLa, HuH-7) and primary tissues (mouse testis, rat liver), providing benchmarks for expected detection levels .

What factors should be considered when comparing results from different CYP51 antibodies?

When comparing results obtained with different CYP51 antibodies, researchers should consider:

• Epitope Differences:

  • The ab210792 antibody targets a C-terminal epitope (amino acids 300 to C-terminus)

  • Other commercial antibodies may target different regions, affecting detection in applications where protein conformation matters

• Production Methods:

  • Polyclonal vs. monoclonal differences in epitope recognition

  • Host species (rabbit vs. mouse) that may affect secondary antibody selection and potential cross-reactivity

• Validation Status:

  • Number of citations supporting antibody use in specific applications

  • Availability of knockout/knockdown validation data

• Cross-Reactivity Profiles:

  • Documented reactivity with related cytochrome P450 family members

  • Species cross-reactivity differences that may affect interpretation in comparative studies

• Application-Specific Performance:

  • Some antibodies perform better in certain applications (WB vs. IHC vs. IP)

  • Buffer compatibility differences that may affect results in specialized protocols

Researchers should document and report the specific antibody catalog numbers, dilutions, and protocols used to ensure experimental reproducibility across laboratories .