HEME1 Antibody

What is HO-1 and what is its biological function?

HO-1 (Heme Oxygenase 1) is an inducible enzyme that catalyzes the oxidative cleavage of heme at the alpha-methene bridge carbon. This reaction releases carbon monoxide (CO) and generates biliverdin IXalpha, while simultaneously releasing the central heme iron chelate as ferrous iron . The enzyme plays a crucial cytoprotective role by affording protection against programmed cell death, primarily through its ability to catabolize free heme and prevent it from sensitizing cells to undergo apoptosis . HO-1 is encoded by the HMOX1 gene, which is regulated by diverse negative and positive transcription factors binding to the upstream regulatory region .

What are the molecular characteristics of HO-1 antibodies used in research?

HO-1 antibodies used in research are typically polyclonal or monoclonal immunoglobulins raised against specific epitopes of the human HMOX1 protein. Commercial antibodies like ab137749 are rabbit polyclonal antibodies generated using immunogens corresponding to recombinant fragment proteins within Human HMOX1 amino acid 1 to C-terminus . These antibodies have a predicted molecular weight detection of approximately 32-33 kDa when used in Western blotting applications . Research-grade antibodies undergo validation for specificity across multiple applications and species, with most HO-1 antibodies demonstrating cross-reactivity with human, mouse, and rat samples .

How are HO-1 protein expression levels altered in pathological conditions?

HO-1 expression is significantly altered during various pathological conditions. During SARS-COV-2 infection, HO-1 promotes SARS-CoV-2 ORF3A-mediated autophagy, though it is unlikely to be required for ORF3A-mediated induction of reticulophagy . In renal cancer tissue, HO-1 is expressed at higher levels compared to normal tissue . The expression of HO-1 is highly inducible by various stressors, particularly free heme, which binds to Bach1 (a repressor), produces reactive oxygen species (ROS), and efficiently induces the expression of HMOX1 . The regulatory mechanism involves the liberation of Nrf2 (a transcription activator) and degradation of Keap1 (a cytosolic protein that binds to Nrf2), allowing Nrf2 to bind to the antioxidant responsive element (ARE) in the HMOX1 promoter .

What are the validated applications for HO-1 antibodies in laboratory research?

HO-1 antibodies have been validated for multiple research applications with specific methodological requirements:

These applications have been validated across multiple species including human, mouse, and rat samples, with cited publications confirming their efficacy in research settings .

How should researchers optimize Western blotting protocols for HO-1 detection?

For optimal Western blot detection of HO-1, researchers should follow these methodological steps:

• Sample preparation: Extract proteins from whole cell lysates or tissue samples, loading approximately 30-50 μg of protein per lane

• Gel electrophoresis: Separate samples using 12% SDS-PAGE for optimal resolution of the 32-33 kDa HO-1 protein

• Transfer: Transfer proteins to nitrocellulose or PVDF membranes using standard protocols

• Blocking: Block membranes with appropriate blocking buffer (typically 5% non-fat milk or BSA)

• Primary antibody incubation: Dilute HO-1 antibody between 1/500 to 1/5000 depending on the specific antibody and expected expression level

• Detection: Use HRP-conjugated secondary antibodies (anti-rabbit IgG for rabbit polyclonal primary antibodies)

This protocol has been validated across multiple cell lines including H1299, HeLa, NIH-3T3, and Raw 264.7 cells, with consistent detection of the predicted 32-33 kDa band .

What is the recommended protocol for immunohistochemistry with HO-1 antibodies?

For immunohistochemical detection of HO-1 in tissue sections, the following methodological approach is recommended:

• Tissue preparation: Use formalin-fixed, paraffin-embedded tissue sections cut at 4-6 μm thickness

• Deparaffinization: Remove paraffin and rehydrate sections using standard protocols

• Antigen retrieval: Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) for 15 minutes

• Endogenous peroxidase blocking: Block endogenous peroxidase activity with hydrogen peroxide

• Antibody dilution: Dilute HO-1 antibody (e.g., ab137749) at 1/100 to 1/500 concentration

• Incubation: Incubate sections with primary antibody at room temperature or 4°C overnight

• Detection: Use appropriate detection systems (e.g., HRP-DAB) to visualize the antibody binding

This protocol has been successfully applied to various tissues including spleen and xenograft models, with specific cytoplasmic localization of HO-1 observed .

How does HO-1 modulate endothelial cell activation in transplant rejection?

HO-1 plays a critical role in modulating endothelial cell (EC) activation during antibody-mediated rejection (AMR) in organ transplantation. Research demonstrates that HLA class I (HLA I) antibodies activate ECs by upregulating proinflammatory adhesion molecules and chemokines, including VCAM-1, ICAM-1, IL-8, and MCP-1 .

The modulatory mechanism of HO-1 involves:

• Pharmacological induction of HO-1 with cobalt-protoporphyrin IX reduces HLA I antibody-dependent upregulation of VCAM-1

• Conversely, inhibition of HO-1 with zinc-protoporphyrin IX or siRNA-mediated knockdown increases HLA I antibody-dependent EC activation

• Carbon monoxide (CO), a gaseous product of HO-1 activity, blocks HLA I antibody-dependent EC activation

• In vitro adhesion assays demonstrate that HO-1 upregulation counteracts HLA I antibody-induced monocyte binding to ECs

These findings suggest that targeted induction of HO-1 may represent a novel therapeutic approach for treating antibody-mediated rejection in solid organ transplantation by limiting endothelial activation and subsequent inflammatory responses .

What is the relationship between heme and antibody production and function?

The relationship between heme and antibody production/function is multifaceted:

• Heme at low concentrations (several μM) enhances specific antibody production in animal models. In experiments with rats, heme-containing formulations significantly increased the levels of anti-BSA IgG and IgM in sera compared to formulations without heme .

• The promotion ability of heme in liposomal formulations (heme+PC or heme+REcL) approximated that of Complete Freund's Adjuvant (CFA), suggesting heme can act as an immune response enhancer .

• Heme interaction with antibodies correlates with specific sequence characteristics of the antigen-binding site and serves as a predictor for several molecular and functional qualities of antibodies, including:

  • Increased hydrophobicity

  • Reduced thermodynamic stability

  • Higher intrinsic polyreactivity

  • Greater propensity for self-binding

  • Reduced expression yields

• These heme-binding qualities are particularly relevant for therapeutic antibody development, as they have been associated with failure of drug candidates to progress through clinical trials .

This relationship suggests that heme not only influences antibody production but can also be used as a predictive tool for assessing antibody qualities essential for therapeutic applications .

How do free heme levels influence the regulation of HMOX1 gene expression?

Free heme regulation of HMOX1 gene expression involves a complex interplay of transcription factors and cellular stress responses:

• Under non-stress conditions, the cytosolic protein Keap1 binds to transcription factor Nrf2, allowing Bach1 (a repressor) to bind to the antioxidant responsive element (ARE) in the HMOX1 promoter, thus suppressing gene expression .

• Under stress conditions or elevated free heme levels, the following mechanisms activate HMOX1 expression:

  • Free heme directly binds to Bach1, causing its dissociation from ARE

  • Heme induces reactive oxygen species (ROS) production

  • ROS lead to liberation of Nrf2 from Keap1 and degradation of Keap1

  • Liberated Nrf2 competes with Bach1 for binding to ARE, promoting HMOX1 expression

  • STAT3 can also bind to STAT-binding element (SBE) to promote HMOX1 expression

• Free heme is particularly efficient at inducing HMOX1 expression through these dual mechanisms of direct Bach1 binding and ROS generation .

This regulatory system allows for rapid induction of HO-1 in response to elevated heme levels, providing a cytoprotective feedback mechanism to prevent heme-induced cellular damage.

What are common challenges when detecting HO-1 in different cellular compartments?

Detecting HO-1 in different cellular compartments presents several technical challenges that researchers should consider:

• Subcellular localization variation: HO-1 can be detected in multiple cellular compartments, including endoplasmic reticulum and nucleus, requiring appropriate fixation and permeabilization protocols .

• Isoform specificity: Both membrane-bound and soluble forms of HO-1 exist, with distinct functional activities. When designing experiments, researchers should consider which form they intend to detect .

• Expression level differences: HO-1 is highly inducible, with basal expression levels varying significantly across cell and tissue types. Experimental designs should include appropriate positive controls and may require treatment with HO-1 inducers to observe detectable expression in some systems .

• Protocol optimization: For immunofluorescence detection of HO-1, 4% paraformaldehyde fixation and optimized permeabilization are critical. For nuclear HO-1 detection, more stringent permeabilization may be required compared to detection of ER-associated HO-1 .

To address these challenges, researchers should validate their HO-1 antibodies using both positive controls (HO-1 inducer-treated cells) and negative controls (HO-1 knockdown cells) to confirm specificity for their specific experimental system.

How can researchers validate the specificity of their HO-1 antibody?

Validating HO-1 antibody specificity requires a multi-faceted approach:

• Multiple-cell line validation: Test the antibody across different cell lines with varying HO-1 expression levels. For example, ab137749 has been validated in multiple cell lines including H1299, HeLa, NIH-3T3, JC, BCL1, C2C12, and Raw 264.7 cells .

• Molecular weight confirmation: Verify that the detected band appears at the predicted molecular weight of 32-33 kDa in Western blot applications .

• Induction experiments: Compare HO-1 detection in untreated versus treated samples. For example, testing antibody reactivity in cells treated with HO-1 inducers (e.g., heme or cobalt-protoporphyrin IX) to confirm increased signal intensity .

• Knockdown/knockout validation: Perform siRNA-mediated knockdown or CRISPR/Cas9 knockout of HO-1 and confirm reduced or absent antibody signal .

• Cross-application validation: Confirm specificity across multiple applications (WB, IHC, ICC/IF) to ensure consistent results across experimental platforms .

• Loading controls: Always include appropriate loading controls in experiments to normalize protein expression and differentiate specific from non-specific signals .

This comprehensive validation approach will ensure that research findings based on HO-1 antibody detection are reliable and reproducible.

How is HO-1 being investigated in therapeutic approaches for transplant rejection?

Current research on HO-1 as a therapeutic target for transplant rejection focuses on several promising approaches:

• Pharmacological HO-1 induction: Studies have demonstrated that induction of HO-1 with cobalt-protoporphyrin IX reduces HLA I antibody-dependent endothelial cell activation, suggesting potential therapeutic applications in preventing antibody-mediated rejection (AMR) .

• Carbon monoxide (CO) delivery: As a product of HO-1 enzymatic activity, CO has shown promise in blocking HLA I antibody-dependent endothelial cell activation. CO-releasing molecules (CORMs) are being investigated as potential therapeutic agents that might mimic the beneficial effects of HO-1 induction without the need for enzyme upregulation .

• Targeted endothelial HO-1 modulation: Research shows that HO-1 specifically modulates endothelial cell responses to HLA I antibodies, including the expression of adhesion molecules (VCAM-1, ICAM-1) and chemokines (IL-8, MCP-1). Targeting endothelial HO-1 induction may provide a more specific approach to preventing AMR in solid organ transplantation .

• Molecular mechanism investigations: Ongoing research is exploring the precise molecular pathways by which HO-1 exerts its protective effects against antibody-mediated endothelial activation, potentially identifying additional therapeutic targets within these pathways .

These approaches represent cutting-edge investigations into HO-1-based therapies for transplant rejection, with potential applications in other antibody-mediated inflammatory conditions.

What does the interaction between antibodies and heme reveal about therapeutic antibody development?

Research on antibody-heme interactions has revealed important insights for therapeutic antibody development:

• Predictive biomarker: Interaction with heme serves as a strong predictor of various molecular and functional qualities of antibodies that are critical for therapeutic applications .

• Sequence-function relationships: Antibodies that interact with heme possess specific sequence traits in their antigen-binding regions, providing valuable information about the relationship between antibody sequence and function .

• Correlations with problematic properties: Heme-binding antibodies demonstrate:

  • Increased hydrophobicity

  • Higher propensity for self-binding

  • Higher intrinsic polyreactivity

  • Reduced expression yields

  • Reduced stability

• Clinical development implications: These qualities have been associated with failure of antibody drug candidates to advance through clinical trials, making heme interaction a potential screening tool for early identification of problematic antibody candidates .

• Analytical tool development: The correlation between heme binding and antibody properties suggests the utility of heme-based assays for the early detection of unwanted properties in candidate therapeutic antibodies .

This research extends the basic understanding of antibody molecules and facilitates the development of convenient analytical assays for screening therapeutic antibody candidates early in the development process, potentially saving significant time and resources in drug development efforts.