HMOX2 Antibody, HRP conjugated

What is HMOX2 and what biological functions does it serve?

HMOX2 (Heme Oxygenase Decycling 2) is a constitutively expressed enzyme in mammalian tissues that plays a crucial role in heme catabolism. Unlike its inducible counterpart HMOX1, HMOX2 is consistently expressed under normal physiological conditions, particularly in the brain and testes. HMOX2 catalyzes the rate-limiting step in heme degradation, converting heme to biliverdin while releasing carbon monoxide (CO) and iron. This enzymatic activity serves multiple physiological functions, including cellular protection against oxidative stress, regulation of vascular tone, and neurotransmission. In the brain, CO produced by HMOX2 functions as a neurotransmitter affecting various physiological processes, making HMOX2 particularly significant in neurobiology . The biological importance of HMOX2 extends to neuroprotection mechanisms and maintenance of cellular homeostasis, with its CO signaling pathway closely paralleling that of nitric oxide . Understanding HMOX2's structure and function is essential for researchers investigating neurodegenerative diseases, oxidative stress responses, and cellular signaling mechanisms.

How do HRP-conjugated HMOX2 antibodies differ from unconjugated versions in research applications?

HRP-conjugated HMOX2 antibodies offer significant advantages over unconjugated versions by eliminating the need for secondary antibody incubations, which streamlines experimental workflows and reduces potential sources of variability. The direct conjugation of horseradish peroxidase to the HMOX2 antibody enables straightforward detection through standard chemiluminescent or chromogenic substrates. This modification is particularly valuable in Western blotting applications, where it allows for rapid development and typically offers enhanced sensitivity compared to two-step detection methods. HRP-conjugated HMOX2 antibodies are available in various formats, including mouse monoclonal antibodies like the B-3 clone (sc-17786) that can detect HMOX2 across human, mouse, and rat samples . When using HRP-conjugated antibodies, researchers should consider how the conjugation might affect antibody binding kinetics, as the attached enzyme may occasionally influence epitope recognition or binding affinity, necessitating protocol optimization compared to unconjugated versions.

What are the confirmed species reactivity profiles for commercially available HMOX2 antibodies?

Commercial HMOX2 antibodies demonstrate variable cross-species reactivity that researchers must consider when designing experiments. Based on available data, most HMOX2 antibodies recognize human HMOX2, with many extending reactivity to mouse and rat orthologs. For instance, the B-3 mouse monoclonal antibody (sc-17786) has confirmed reactivity with mouse, rat, and human HMOX2 . Similarly, the rabbit polyclonal antibody (ABIN673809) targeting amino acids 31-150 demonstrates robust cross-reactivity with human, mouse, and rat HMOX2 . The AF3170 antibody shows validated reactivity with endogenous HMOX2 in human cell lines (Jurkat and HeLa), mouse cell lines (C2C12 and DA3), and rat cell lines (PC-12), as confirmed by Western blot analysis . These cross-species validations are particularly valuable for comparative studies across model organisms. Some HMOX2 antibodies even demonstrate broader reactivity profiles extending to monkey, hamster, dog, pig, cow, crayfish, guinea pig, and sheep samples . Researchers should carefully review species validation data when selecting an appropriate HMOX2 antibody for their specific experimental system.

What are the optimal sample preparation techniques for detecting HMOX2 using HRP-conjugated antibodies?

Optimal sample preparation for HMOX2 detection using HRP-conjugated antibodies requires careful consideration of several critical factors. For cellular samples, lysis buffers containing 1% Triton X-100 or RIPA buffer supplemented with protease inhibitors help preserve HMOX2 integrity while efficiently extracting the protein. When working with tissue samples, homogenization in cold lysis buffer followed by sonication improves protein extraction efficiency. The demonstrated detection of HMOX2 in various cell lines, including Jurkat human acute T cell leukemia, HeLa human cervical epithelial carcinoma, C2C12 mouse myoblast, DA3 mouse myeloma, and PC-12 rat adrenal pheochromocytoma cell lines, confirms the versatility of these preparation methods . Based on published Western blot protocols, researchers should load 15-20 μg of total protein per well for cell lines and 30-40 μg for tissue homogenates to achieve optimal signal-to-noise ratios. Notably, HMOX2 typically appears at approximately 36-38 kDa in standard SDS-PAGE systems, while in capillary-based Simple Western systems, it migrates at approximately 43 kDa . This molecular weight discrepancy between different detection platforms should be considered when interpreting results.

How should researchers optimize Western blotting protocols specifically for HMOX2 detection using HRP-conjugated antibodies?

Western blotting protocols for HMOX2 detection using HRP-conjugated antibodies require specific optimization strategies to enhance sensitivity and specificity. Based on published methodologies, researchers should use PVDF membranes rather than nitrocellulose due to PVDF's superior protein binding capacity and compatibility with HRP detection systems . Blocking solutions containing 5% non-fat dry milk in TBST (Tris-buffered saline with 0.1% Tween-20) typically provide optimal results, though BSA-based blocking may be preferable when phospho-specific detection is necessary in multiplexed experiments. For HRP-conjugated HMOX2 antibodies, dilution ratios between 1:500 and 1:2000 are recommended, with overnight incubation at 4°C generally yielding better results than shorter incubations at room temperature. When working with mouse monoclonal HMOX2 antibodies conjugated to HRP (such as the B-3 clone), researchers should consider using m-IgG Fc BP-HRP bundles to minimize background from endogenous immunoglobulins in tissue samples . For signal development, enhanced chemiluminescence (ECL) substrates provide excellent sensitivity, with exposure times typically ranging from 30 seconds to 5 minutes depending on HMOX2 expression levels in the sample.

What validation strategies should researchers employ to confirm HMOX2 antibody specificity?

Rigorous validation of HMOX2 antibody specificity is essential for generating reliable research data. A comprehensive validation approach should include multiple complementary strategies. First, researchers should perform side-by-side comparison of HMOX2 detection in wild-type samples versus HMOX2 knockout or knockdown samples where possible. Second, peptide competition assays can confirm specificity by pre-incubating the HMOX2 antibody with excess immunizing peptide before application to samples; a specific antibody will show significantly reduced or eliminated signal. Third, researchers should verify that detected bands correspond to the expected molecular weight of HMOX2 (approximately 36-38 kDa in standard Western blots or 43 kDa in capillary-based systems) . Fourth, comparing results across multiple antibodies targeting different HMOX2 epitopes can further validate specificity; for instance, comparing results between the mouse monoclonal antibody recognizing full-length HMOX2 (AA 1-316) and the rabbit polyclonal antibody targeting AA 31-150 . Fifth, immunoprecipitation followed by mass spectrometry can provide definitive confirmation of antibody specificity. Finally, researchers should be mindful of potential cross-reactivity with HMOX1, the inducible isoform, which shares approximately 43% sequence identity with HMOX2 but differs in size (32 kDa versus 36 kDa).

How can researchers effectively use HMOX2 antibody, HRP conjugated, in immunohistochemistry and immunofluorescence applications?

HMOX2 antibody, HRP conjugated, can be effectively utilized in immunohistochemistry (IHC) and immunofluorescence (IF) applications with appropriate protocol optimization. For IHC applications, antigen retrieval is crucial; heat-induced epitope retrieval using citrate buffer (pH 6.0) typically yields optimal results for HMOX2 detection in paraffin-embedded tissues. The B-3 monoclonal antibody (sc-17786) has demonstrated effectiveness in both paraffin-embedded and frozen section IHC protocols . When using HRP-conjugated antibodies for IHC, researchers should be particularly attentive to endogenous peroxidase activity, which can be effectively quenched using 0.3% hydrogen peroxide in methanol for 15-30 minutes prior to antibody incubation. For immunofluorescence applications, HMOX2 antibodies have been successfully employed in both cultured cells (IF-cc) and paraffin-embedded tissue sections (IF-p) . When using HRP-conjugated antibodies for IF, tyramide signal amplification systems can convert the HRP activity into robust fluorescent signal with significantly improved sensitivity compared to conventional fluorophore-conjugated secondary antibodies. Optimal dilutions for IF typically range from 1:50 to 1:200, depending on the specific antibody and sample type, with overnight incubation at 4°C recommended for maximum sensitivity and minimal background.

What controls should be included when performing experiments with HMOX2 antibody, HRP conjugated?

A robust experimental design with HMOX2 antibody, HRP conjugated, necessitates multiple controls to ensure data validity and interpretability. Researchers should include both positive and negative controls in every experiment. Positive controls should incorporate samples with known HMOX2 expression; based on published data, Jurkat human acute T cell leukemia, HeLa human cervical epithelial carcinoma, C2C12 mouse myoblast, DA3 mouse myeloma, and PC-12 rat adrenal pheochromocytoma cell lines all express detectable levels of HMOX2 and serve as excellent positive controls . Negative controls should include samples known to lack HMOX2 expression or, ideally, HMOX2 knockout samples. For antibody specificity controls, researchers should perform parallel experiments using isotype-matched irrelevant antibodies (for monoclonal antibodies) or pre-immune serum (for polyclonal antibodies). When working with HRP-conjugated antibodies specifically, substrate-only controls are essential to assess potential endogenous peroxidase activity or non-specific substrate reactions. For quantitative experiments, researchers should include calibration controls with recombinant HMOX2 protein at known concentrations to establish a standard curve. Finally, technical replicates (minimum of three) and biological replicates (derived from independent samples) are necessary to establish statistical significance and ensure reproducibility.

How does HMOX2 detection differ between various cell and tissue types?

HMOX2 detection profiles exhibit significant variations across different cell and tissue types, reflecting both biological differences in expression and technical considerations in sample preparation. At the tissue level, HMOX2 is most abundantly expressed in the brain, testes, and endothelial cells, with moderate expression in the liver, kidney, and spleen. When comparing detection across cell lines, Western blot and Simple Western analyses have demonstrated successful HMOX2 detection in diverse cell types including Jurkat human acute T cell leukemia, HeLa human cervical epithelial carcinoma, C2C12 mouse myoblast, DA3 mouse myeloma, and PC-12 rat adrenal pheochromocytoma cells . These diverse cell types may require different lysis conditions for optimal HMOX2 extraction; neural tissues often benefit from stronger detergent formulations due to their high lipid content. The apparent molecular weight of HMOX2 remains consistent at approximately 36-38 kDa in standard Western blots across most cell types, though post-translational modifications may occasionally cause slight variations in migration patterns. In capillary-based Simple Western systems, HMOX2 typically migrates at approximately 43 kDa . Intracellular localization patterns also vary; while primarily cytoplasmic in most cell types, HMOX2 can associate with the endoplasmic reticulum and occasionally demonstrate nuclear localization in certain contexts, which may influence detection efficiency in immunocytochemistry applications.

What are common technical challenges when using HMOX2 antibody, HRP conjugated, and how can they be addressed?

Researchers frequently encounter several technical challenges when working with HMOX2 antibody, HRP conjugated, each requiring specific troubleshooting approaches. High background signal is a common issue, typically resulting from inadequate blocking or excessive antibody concentration. This can be addressed by increasing blocking time (minimum 1 hour at room temperature), using alternative blocking agents (switching between milk and BSA-based blockers), and further diluting the HRP-conjugated antibody. Weak or absent HMOX2 signal may stem from inefficient protein extraction or transfer; optimize by using stronger lysis buffers containing 1% SDS for challenging samples and extend transfer times for PVDF membranes to 90-120 minutes at 100V. Multiple bands in Western blots may indicate degradation products, cross-reactivity, or post-translational modifications of HMOX2; address by adding additional protease inhibitors during sample preparation and confirming specificity through knockout/knockdown controls. Non-specific binding can be reduced by including 0.1-0.3% Tween-20 in wash buffers and optimizing antibody dilutions. Signal variability between replicates often stems from inconsistent sample loading or transfer efficiency; implement loading controls (β-actin, GAPDH) and consider Ponceau S staining of membranes post-transfer to verify uniform protein transfer before antibody incubation.

How can researchers quantitatively analyze HMOX2 expression using HRP-conjugated antibodies?

Quantitative analysis of HMOX2 expression using HRP-conjugated antibodies requires careful attention to several methodological aspects to ensure accuracy and reproducibility. For Western blot quantification, researchers should capture images within the linear detection range of their imaging system, avoiding over-exposed bands that lead to signal saturation and inaccurate quantification. Densitometric analysis using software like ImageJ, Image Lab, or similar platforms should include normalization to appropriate housekeeping proteins (β-actin, GAPDH, or β-tubulin) to account for loading variations. When analyzing HMOX2 across multiple samples, include a calibration sample on each blot to allow for inter-blot normalization. For more precise quantification, researchers can employ the Simple Western system, which has demonstrated reliable detection of HMOX2 at approximately 43 kDa across human, mouse, and rat samples using 5 μg/mL of HMOX2 antibody . This capillary-based immunoassay system offers superior reproducibility and wider dynamic range compared to traditional Western blotting. For absolute quantification, researchers should establish a standard curve using purified recombinant HMOX2 protein at known concentrations. When analyzing HMOX2 in tissue sections via IHC, quantitative image analysis using specialized software can assess staining intensity and distribution, though careful standardization of staining protocols and imaging parameters is essential for inter-sample comparisons.

How is HMOX2 antibody, HRP conjugated, being utilized in neurodegenerative disease research?

HMOX2 antibody, HRP conjugated, has become an invaluable tool in neurodegenerative disease research due to HMOX2's critical role in neuroprotection and cellular stress responses. Researchers are applying these antibodies to investigate alterations in HMOX2 expression and localization across various neurodegenerative conditions, including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS). The constitutive expression of HMOX2 in the brain, where carbon monoxide functions as a neurotransmitter influencing various physiological processes, makes it particularly relevant to neurological research . HRP-conjugated HMOX2 antibodies enable efficient protein detection in both human post-mortem brain samples and animal models of neurodegeneration, facilitating direct quantitative comparisons of HMOX2 levels between diseased and healthy tissues. In neurodegenerative disease models, researchers are using these antibodies to examine how HMOX2-mediated heme metabolism and subsequent biliverdin and free iron generation contribute to cellular antioxidant defense mechanisms when faced with pathological protein aggregation and oxidative stress . The ability to detect HMOX2 in both mouse and rat samples using the same antibody (as demonstrated with several commercially available antibodies) allows for translational research connecting animal model findings to human disease mechanisms.

What recent advances have emerged in studying HMOX2 in cellular stress responses using HRP-conjugated antibodies?

Recent research utilizing HRP-conjugated HMOX2 antibodies has significantly advanced our understanding of HMOX2's role in cellular stress responses. Unlike its inducible counterpart HMOX1, HMOX2 is constitutively expressed but nonetheless plays critical roles in oxidative stress adaptation through baseline heme catabolism and carbon monoxide production. Researchers have utilized Western blotting with HRP-conjugated HMOX2 antibodies to demonstrate that while HMOX2 protein levels remain relatively stable under stress conditions, its subcellular localization and enzymatic activity undergo significant changes. These studies have been facilitated by the availability of antibodies that effectively detect HMOX2 across multiple species, including human, mouse, and rat samples, as validated through Western blot analyses in various cell lines . Investigations into HMOX2's interaction networks have revealed its cooperation with various stress-response pathways, including Nrf2 signaling and mitochondrial quality control mechanisms. The application of HRP-conjugated antibodies in immunoprecipitation followed by mass spectrometry has identified novel HMOX2 binding partners that modulate its activity during cellular stress. Additionally, recent work comparing HMOX2 function across different tissues has highlighted tissue-specific roles in stress adaptation, with particularly important functions in the brain and cardiovascular system, where its enzymatic products (carbon monoxide, biliverdin, and iron) influence both physiological and pathological processes.

What emerging applications exist for HMOX2 antibody, HRP conjugated, in cancer and vascular research?

Emerging applications for HMOX2 antibody, HRP conjugated, in cancer and vascular research are expanding our understanding of this enzyme's role in complex disease processes. In cancer research, HMOX2 antibodies are being increasingly utilized to investigate the constitutive expression patterns of this enzyme across various tumor types compared to their non-malignant counterparts. While HMOX1 has been more extensively studied in oncology due to its stress-inducible nature, recent evidence suggests HMOX2 may have distinct functions in tumorigenesis and therapy resistance. Western blot analysis using HRP-conjugated HMOX2 antibodies has demonstrated variable expression levels across different cancer cell lines, including Jurkat human acute T cell leukemia cells and HeLa human cervical epithelial carcinoma cells . In vascular research, HMOX2's constitutive expression in endothelial cells makes it a key regulator of vascular tone through carbon monoxide production. Researchers are applying immunohistochemistry with HRP-conjugated HMOX2 antibodies to investigate its expression patterns in vascular pathologies including atherosclerosis, hypertension, and diabetic vasculopathies. The ability of certain HMOX2 antibodies to cross-react with samples from multiple species, including human, mouse, rat, and other mammals , facilitates translational research connecting animal models to human vascular disease. Furthermore, multiplex immunofluorescence approaches incorporating HMOX2 antibodies are revealing its co-localization patterns with other key vascular signaling molecules, providing insights into its regulatory networks in vascular homeostasis and pathology.