CYP27B1 Antibody, HRP conjugated

What is CYP27B1 and what is its biological significance?

CYP27B1 (Cytochrome P450 family 27 subfamily B polypeptide 1) is a critical enzyme in vitamin D metabolism that catalyzes the conversion of 25-hydroxyvitamin D3 (25(OH)D) to the biologically active form 1-alpha,25-dihydroxyvitamin D3 (1,25(OH)2D). This 508-amino acid protein belongs to the XXVIIB subfamily of the cytochrome P450 family and primarily localizes to the mitochondrion . It plays an essential role in calcium metabolism, bone development, and various physiological processes . Also known by numerous synonyms including 1alpha(OH)ase, 25-hydroxyvitamin D-1 alpha hydroxylase, and VD3 1A hydroxylase, CYP27B1 is predominantly expressed in the kidney but has been detected in various other tissues as well . Alterations in CYP27B1 expression and activity have been implicated in multiple pathological conditions, including breast cancer progression and vascular calcification .

What are the key specifications of CYP27B1 Antibody, HRP conjugated for research applications?

CYP27B1 Antibody, HRP conjugated is a polyclonal antibody derived from rabbit hosts, typically using KLH-conjugated synthetic peptides from human CYP27B1 as the immunogen . The horseradish peroxidase (HRP) conjugation enables direct detection without secondary antibodies in various applications. These antibodies are generally provided at a concentration of 1μg/μl in an aqueous buffered solution containing 0.01M TBS (pH 7.4) with 1% BSA, 0.03% Proclin300, and 50% Glycerol . The antibody targets specific epitopes within the 1-100/508 amino acid range of the CYP27B1 protein . Commercial preparations are purified using Protein A affinity chromatography to ensure specificity and optimal performance in experimental applications . For research integrity, proper storage at -20°C with aliquoting to prevent repeated freeze-thaw cycles is essential for maintaining antibody activity and specificity .

What are the validated applications for CYP27B1 Antibody, HRP conjugated?

CYP27B1 Antibody, HRP conjugated has been validated for multiple research applications including Western blotting (WB), enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry on paraffin-embedded tissues (IHC-P) . The antibody demonstrates primary reactivity with mouse CYP27B1 and predicted cross-reactivity with human, rat, dog, cow, sheep, pig, and horse species based on sequence homology . For immunohistochemical applications, researchers typically use the antibody at dilutions ranging from 1:75 to 1:200 with incubation times varying from 60 minutes to overnight at 4°C, depending on the specific protocol and tissue type . In Western blot analysis, the antibody can detect both native and modified forms of CYP27B1, including potential glycosylated variants that may play roles in pathological conditions . For optimal results, antigen retrieval methods (typically 30 minutes) are recommended when working with formalin-fixed tissues .

How should immunohistochemical analysis be optimized for CYP27B1 detection?

For optimal immunohistochemical detection of CYP27B1, tissues should be fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS), followed by dehydration in ascending ethanol series, paraffin embedding, and sectioning at 2 μm thickness . Prior to antibody application, endogenous peroxidase activity should be neutralized with 3% H₂O₂ for 10 minutes, followed by blocking non-specific binding with 1% bovine serum albumin (BSA) in PBS . For CYP27B1 detection, polyclonal goat anti-CYP27B1 antibody at 1:200 dilution is recommended with overnight incubation at 4°C . Secondary detection systems typically involve biotin-conjugated rabbit anti-goat IgG followed by treatment with peroxidase-streptavidin complex . Visualization is accomplished using 0.03% 3'3-diaminobenzidine tetrahydrochloride (DAB) solution containing 0.001% H₂O₂, 10 mM Imidazole, and 50 mM Trizma hydrochloride, with Mayer's Hematoxylin Solution for nuclear counterstaining . When investigating CYP27B1 in potentially calcified tissues, parallel sections should be evaluated with von Kossa staining to identify calcium deposits and assess co-localization .

What controls and validation methods are essential when working with CYP27B1 antibodies?

When working with CYP27B1 antibodies, multiple validation approaches are necessary to ensure specificity and reliability of results. Essential controls include peptide neutralization assays, where the antibody is pre-incubated with a 5-fold excess (by weight) of the specific CYP27B1 peptide antigen for 2 hours at room temperature before application to samples . Negative controls should include normal IgG from the same species as the primary antibody (e.g., normal goat IgG at appropriate dilution) . For tissues with potential calcification that might cause non-specific binding, decalcification controls are crucial—these involve treating parallel sections with 0.6 N hydrochloric acid for 24 hours at room temperature prior to immunostaining . The effectiveness of decalcification should be verified using von Kossa staining . Western blot validation should demonstrate the correct molecular weight band for CYP27B1, with parallel blots using peptide-neutralized antibody showing absence of this band . For glycosylated CYP27B1 variants, additional verification using Periodic acid-Schiff (PAS) staining is recommended to confirm glycoprotein status .

How does CYP27B1 expression differ between normal and pathological tissues?

In pathological conditions like ectopic calcification, CYP27B1 shows distinct upregulation and co-localization with calcified lesions . In animal models of hyperphosphatemia (kl/kl mice), CYP27B1 protein is highly expressed and co-localizes with von Kossa-positive calcified regions in kidney cortex arteries and aorta . This suggests a potential role for CYP27B1 in the pathogenesis of vascular calcification, particularly under conditions of hyperphosphatemia .

What is the relationship between CYP27B1 expression and ectopic calcification in vascular tissues?

The relationship between CYP27B1 expression and ectopic calcification in vascular tissues appears to be mechanistically significant, particularly under hyperphosphatemic conditions. Research using kl/kl mouse models has demonstrated that CYP27B1 protein expression is markedly upregulated in tissues undergoing ectopic calcification . Immunohistochemical analyses reveal distinct co-localization of CYP27B1 with von Kossa-positive calcified lesions in kidney cortex arteries and aorta of these mice . This co-expression pattern suggests that CYP27B1 may play a direct role in the calcification process rather than being merely a consequence of it. Importantly, when vascular smooth muscle cells (VSMCs) are subjected to hyperphosphatemic conditions, they exhibit increased CYP27B1 expression that correlates with calcification progression . The persistence of CYP27B1 immunoreactivity in these lesions even after decalcification treatment confirms that the observed CYP27B1 staining is not an artifact of non-specific binding to calcium deposits . These findings suggest that extra-renal expression of CYP27B1, particularly in its glycosylated form, may be a prerequisite for ectopic calcification of VSMCs under hyperphosphatemic conditions, potentially through local modification of vitamin D metabolism .

How can researchers distinguish between different post-translational modifications of CYP27B1?

Distinguishing between different post-translational modifications of CYP27B1 requires a multi-analytical approach combining immunodetection with specific biochemical assays. For glycosylation analysis, researchers should implement Periodic acid-Schiff (PAS) staining in conjunction with Western blot analysis . This combination allows visualization of glycosylated CYP27B1 variants that may exhibit altered molecular weights compared to the unmodified protein. When analyzing tissue samples potentially containing both native and modified CYP27B1, separating proteins on 12% SDS-polyacrylamide gels provides optimal resolution of different molecular weight forms . For detection of phosphorylation or other modifications, specialized antibodies targeting specific modification sites would be necessary, though these were not specifically mentioned in the provided search results.

To confirm the identity of modified CYP27B1 bands, researchers should employ peptide competition assays where the antibody is pre-incubated with excess CYP27B1 peptide antigen, which should eliminate specific bands . Additionally, mass spectrometry analysis following immunoprecipitation can precisely identify modification types and sites. For tissues with potential calcium-protein interactions, parallel experiments with decalcified and non-decalcified samples are essential to distinguish authentic CYP27B1 signal from potential artifacts due to calcium binding . This comparative approach helps researchers accurately characterize the diverse post-translational states of CYP27B1 that may have distinct functional implications in different physiological and pathological contexts.

What methodological challenges exist in studying the extra-renal expression of CYP27B1?

Studying extra-renal expression of CYP27B1 presents several significant methodological challenges. First, distinguishing true CYP27B1 signals from non-specific binding in tissues with calcification requires careful experimental design . Researchers must employ decalcification controls by treating parallel tissue sections with 0.6 N hydrochloric acid for 24 hours followed by von Kossa staining to confirm removal of calcium deposits before immunohistochemistry . Second, the potentially lower expression levels of CYP27B1 in extra-renal tissues compared to kidney necessitates highly sensitive detection methods and careful optimization of antibody concentrations and incubation times .

Third, the possible presence of tissue-specific post-translational modifications (such as glycosylation) may alter antibody recognition, requiring Western blot analysis with appropriate controls to identify different molecular weight variants . Fourth, co-localization studies to identify cell types expressing CYP27B1 in heterogeneous tissues require double immunostaining techniques with cell-specific markers (such as NaPi-IIa for proximal tubules) . Finally, when studying CYP27B1 in pathological contexts like cancer or vascular calcification, researchers must carefully design scoring systems appropriate for the specific tissue type and pathology . For instance, in breast tissue studies, investigators have had to develop custom evaluation methods due to the lack of previously established protocols for CYP27B1 immunohistochemical assessment in these tissues .

What insights does current research provide about the functional significance of CYP27B1 in vitamin D signaling disruption during cancer progression?

Current research indicates that CYP27B1 plays a complex role in vitamin D signaling disruption during cancer progression, particularly in breast cancer. Studies examining breast cancer tissues have revealed alterations in the vitamin D metabolic and signaling pathways that appear to favor tumor progression . In benign breast lesions, CYP27B1 expression is detected in approximately 55.8% of cases, with relatively consistent expression across most benign conditions (55-56%) except for atypical ductal hyperplasia, which shows reduced expression (36.4%) . This differential expression pattern suggests that changes in CYP27B1 expression may occur early in the neoplastic transformation process.

The relationship between CYP27B1 and other components of the vitamin D pathway, including the vitamin D receptor (VDR) and the catabolic enzyme CYP24A1, appears to be disrupted in malignant transformation . While VDR expression remains high in benign breast lesions (93.5% positivity), CYP24A1 expression is limited to only 19% of these same lesions . This imbalance between synthetic and catabolic vitamin D enzymes may affect local concentrations of active vitamin D metabolites within the tissue microenvironment. Researchers have concluded that "there is a deregulation of the Vitamin D signalling and metabolic pathways in breast cancer, favouring tumour progression" . This suggests that alterations in CYP27B1 function, possibly in conjunction with changes in other vitamin D pathway components, may contribute to creating a microenvironment permissive to cancer development and progression.

What are the optimal Western blot protocols for detecting CYP27B1 using HRP-conjugated antibodies?

For optimal Western blot detection of CYP27B1 using HRP-conjugated antibodies, tissues should first be homogenized in lysis buffer containing 1% Triton X-100, 50 mM Tris-HCl, 150 mM NaCl, and 5 mM EDTA, followed by centrifugation at 12,000 rpm for 10 minutes after a 10-minute incubation on ice . Protein quantification using Bradford assay ensures equal loading across samples . Samples should be denatured in 2× SDS sample buffer and separated on 12% SDS-polyacrylamide gels to achieve optimal resolution of the CYP27B1 protein . Following electrophoresis, proteins should be transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon-P) .

Blocking should be performed with 5% non-fat dried milk in PBS containing 0.05% Tween-20 (PBS-t) for 1 hour at room temperature . For direct detection with HRP-conjugated CYP27B1 antibodies, incubation should occur overnight at 4°C at an optimized dilution (typically 1:200 to 1:400), followed by three washes with PBS-t . Signal detection should utilize enhanced chemiluminescence (ECL) Plus system with appropriate X-ray film exposure . Critical controls must include β-actin (1:3,000 dilution) as a loading control, as well as peptide neutralization assays where the CYP27B1 antibody is pre-incubated with a 5-fold excess of specific CYP27B1 peptide antigen for 2 hours before application to blots . This comprehensive protocol ensures specific and sensitive detection of CYP27B1 protein across diverse experimental conditions.

What strategies can improve the specificity of CYP27B1 detection in challenging tissue samples?

Improving specificity of CYP27B1 detection in challenging tissue samples requires multiple validation strategies and controls. For tissues with potential calcification, which may cause non-specific antibody binding, researchers should perform parallel experiments on decalcified sections treated with 0.6 N hydrochloric acid for 24 hours at room temperature . The effectiveness of decalcification should be confirmed using von Kossa staining before proceeding with immunohistochemistry . Comparing CYP27B1 immunostaining patterns between decalcified and non-decalcified sections helps distinguish authentic signals from artifacts .

Antibody specificity should be rigorously validated through peptide neutralization assays, where the primary antibody is pre-incubated with excess CYP27B1 peptide antigen (5-fold excess by weight is recommended) for 2 hours before application to tissue sections . This competitive inhibition approach should eliminate specific staining while leaving any non-specific binding unaffected. Negative control experiments using normal IgG from the same species as the primary antibody at equivalent concentrations are essential for identifying background staining .

For tissues with high autofluorescence or endogenous peroxidase activity, additional blocking steps may be necessary, including treatment with 3% H₂O₂ for 10 minutes to quench endogenous peroxidases . When investigating co-localization of CYP27B1 with other markers or structures, double-staining techniques with appropriate controls should be employed. For example, co-staining with NaPi-IIa can help identify proximal tubule cells in kidney sections . These comprehensive approaches significantly enhance the specificity and reliability of CYP27B1 detection across diverse experimental contexts.

What emerging research questions are being explored regarding the role of CYP27B1 in ectopic calcification disorders?

Emerging research is focusing on several critical questions regarding CYP27B1's role in ectopic calcification disorders. A primary area of investigation concerns the molecular mechanisms by which extra-renal CYP27B1 expression contributes to pathological calcification processes, particularly in vascular smooth muscle cells under hyperphosphatemic conditions . Research suggests that glycosylated forms of CYP27B1 may be specifically required for ectopic calcification, opening questions about how post-translational modifications alter enzyme function in pathological contexts .

Researchers are also exploring the potential bidirectional relationship between CYP27B1 expression and calcification—investigating whether CYP27B1 upregulation precedes and drives calcification or whether it represents a compensatory response to initial calcium deposition . The co-localization of CYP27B1 with calcified lesions in various tissues raises questions about tissue-specific regulatory mechanisms that may differentially control CYP27B1 expression and activity outside the kidney . Additional research questions include the potential therapeutic implications of targeting extra-renal CYP27B1 to prevent or treat vascular calcification, and the possible interaction between CYP27B1 and other vitamin D metabolic enzymes in creating microenvironments that promote mineral deposition . These investigations may ultimately lead to novel diagnostic markers and therapeutic approaches for calcification disorders, which represent significant health burdens in aging populations and patients with chronic kidney disease.

How might advances in antibody technology improve CYP27B1 research methodologies?

Advances in antibody technology hold significant promise for enhancing CYP27B1 research methodologies across multiple dimensions. The development of monoclonal antibodies with enhanced specificity for distinct CYP27B1 epitopes would allow more reliable detection across diverse experimental conditions, potentially reducing the false positives that can occur with current polyclonal antibodies . Antibodies engineered to recognize specific post-translational modifications of CYP27B1, such as various glycosylation patterns, would enable researchers to differentiate between functional variants of the enzyme and investigate their distinct roles in normal and pathological processes .

Multiplex-compatible antibodies that maintain specificity when used in combination with antibodies against other vitamin D pathway components would facilitate comprehensive analysis of pathway interactions within the same tissue sample . Development of antibodies optimized for super-resolution microscopy techniques would allow precise subcellular localization of CYP27B1, potentially revealing previously unrecognized functional domains within cells . The creation of antibody-based biosensors capable of detecting CYP27B1 enzymatic activity in real-time within living cells or tissues would transform understanding of the dynamic regulation of vitamin D metabolism . Finally, standardized antibody validation protocols specifically designed for CYP27B1 would enhance comparability across studies and accelerate research progress. These technological advancements would collectively address many of the methodological challenges currently limiting CYP27B1 research and potentially reveal new insights into its diverse biological functions.