CHX25 Antibody

What are the key characteristics of the CH25H antibody (1G8)?

CH25H Antibody (1G8) is a mouse monoclonal IgG2b kappa light chain antibody specifically designed for detecting CH25H protein across multiple species including mouse, rat, and human origins . This antibody is available in non-conjugated format and demonstrates reliable detection capabilities through several experimental techniques including western blotting (WB), immunoprecipitation (IP), and enzyme-linked immunosorbent assay (ELISA) . The antibody recognizes the full protein that catalyzes cholesterol conversion to 25-hydroxycholesterol, making it valuable for studying lipid metabolism regulatory pathways .

What immunodetection techniques are compatible with CH25H antibody?

The CH25H Antibody (1G8) has been validated for multiple immunodetection techniques commonly used in research settings:

*Specific dilutions should be optimized based on sample type and experimental conditions .

How should CH25H antibody samples be stored and handled?

Proper storage and handling of CH25H antibody is essential for maintaining its reactivity and specificity. While specific storage conditions for CH25H antibody (1G8) are not detailed in the search results, monoclonal antibodies generally require storage at -20°C for long-term preservation and 4°C for short-term use. Repeated freeze-thaw cycles should be avoided to prevent degradation of the antibody. For handling, aseptic techniques should be employed to prevent contamination, and appropriate personal protective equipment should be used when working with the antibody.

What are the optimal protocols for using CH25H antibody in western blotting experiments?

When employing CH25H antibody for western blotting, researchers should consider the following methodological approach:

• Sample Preparation: Prepare protein lysates from cells or tissues expressing CH25H, ensuring complete lysis and protein solubilization.

• Protein Separation: Separate proteins via SDS-PAGE using an appropriate percentage gel (typically 10-12% for the 272 amino acid CH25H protein).

• Transfer: Transfer proteins to a PVDF or nitrocellulose membrane using standard transfer techniques.

• Blocking: Block non-specific binding sites with 5% non-fat dry milk or BSA in TBST.

• Primary Antibody Incubation: Dilute CH25H antibody (1G8) appropriately (starting with manufacturer recommendations, typically 1:500) and incubate overnight at 4°C.

• Secondary Antibody: Use an appropriate HRP-conjugated anti-mouse IgG secondary antibody.

• Detection: Visualize using enhanced chemiluminescence.

• Controls: Include positive control samples known to express CH25H and negative controls lacking CH25H expression to validate specificity.

The CH25H antibody's mouse monoclonal IgG2b kappa light chain characteristics should be considered when selecting appropriate secondary antibodies and blocking reagents to minimize background interference .

How can researchers validate the specificity of CH25H antibody in their experimental system?

Validating antibody specificity is crucial for generating reliable research data. For CH25H antibody, researchers should implement multiple validation strategies:

• Knockout/Knockdown Controls: Compare antibody reactivity in wild-type samples versus CH25H knockout or knockdown samples.

• Immunoprecipitation Followed by Mass Spectrometry: Confirm that immunoprecipitated proteins include CH25H.

• Peptide Competition Assay: Pre-incubate the antibody with purified CH25H protein or peptide before immunodetection to demonstrate signal suppression.

• Cross-Species Reactivity Testing: Verify reactivity across mouse, rat, and human samples as claimed by the manufacturer .

• Multiple Antibody Concordance: Compare results with alternative CH25H antibodies recognizing different epitopes.

• Recombinant Expression Systems: Test antibody reactivity against recombinant CH25H expressed in suitable cellular systems.

These validation approaches ensure that experimental findings accurately reflect CH25H biology rather than non-specific interactions or artifacts.

What analytical considerations should be addressed when studying CH25H's role in lipid metabolism?

When investigating CH25H's role in lipid metabolism, researchers should consider these analytical factors:

• Oxysterol Measurement: Implement liquid chromatography-mass spectrometry (LC-MS) techniques to accurately quantify 25-hydroxycholesterol levels as the primary enzymatic product of CH25H activity.

• SREBP Processing Analysis: Employ gel-shift assays and western blotting to monitor SREBP cleavage and nuclear translocation, which is directly influenced by CH25H activity .

• Cellular Localization: Use immunofluorescence microscopy with CH25H antibody to confirm endoplasmic reticulum localization where lipid metabolism regulation occurs.

• Metabolic Flux Analysis: Track isotope-labeled cholesterol to measure conversion rates to 25-hydroxycholesterol.

• Cofactor Analysis: Evaluate the diiron center integrity and activity through spectroscopic techniques, as this cofactor is essential for CH25H enzymatic function .

• Transcriptional Effects: Measure expression of genes regulated by oxysterols using qPCR or RNA-seq.

Such comprehensive analytical approaches ensure robust characterization of CH25H's metabolic functions and regulatory roles.

How can CH25H antibody be employed in studying inflammatory responses?

Recent research suggests CH25H and its product 25-hydroxycholesterol play roles in immunomodulation and inflammatory responses. When investigating these aspects, researchers should:

• Cytokine Profiling: Measure changes in pro-inflammatory and anti-inflammatory cytokines in relation to CH25H expression.

• Macrophage Polarization Analysis: Use CH25H antibody in combination with macrophage markers to assess correlation between CH25H expression and macrophage phenotypes.

• Infection Models: Employ CH25H antibody in tissues responding to viral or bacterial infection to track expression changes.

• Signal Transduction Studies: Combine CH25H detection with phosphorylation-specific antibodies to map inflammatory signaling pathways.

• Tissue-Specific Expression Analysis: Quantify CH25H levels across different tissues during inflammation using immunohistochemistry with the CH25H antibody (1G8).

The methodological approach should include appropriate controls and time-course analyses to capture the dynamic nature of inflammatory responses.

What are the key considerations when designing experiments to study CH25H in different tissue types?

When designing experiments to investigate CH25H across different tissues, researchers should address:

• Tissue-Specific Expression Levels: Baseline CH25H expression varies significantly between tissues, requiring adjustment of antibody concentrations and detection methods accordingly.

• Extraction Protocols: Optimize protein extraction based on tissue type, especially for lipid-rich tissues where CH25H is often abundant.

• Fixation Methods for Immunohistochemistry: Evaluate whether paraformaldehyde or other fixatives better preserve CH25H epitope recognition by the antibody.

• Autofluorescence Considerations: Implement appropriate controls and quenching techniques when using fluorescent detection in tissues with high autofluorescence.

• Correlation with Lipid Content: Consider parallel analysis of tissue cholesterol and 25-hydroxycholesterol content to correlate with CH25H expression.

• Species Differences: Although CH25H antibody (1G8) recognizes mouse, rat, and human proteins , account for potential species-specific differences in expression patterns and regulation.

A comprehensive experimental design should incorporate these variables to ensure reliable cross-tissue comparisons of CH25H expression and function.

How should researchers address potential cross-reactivity issues with CH25H antibody?

To mitigate cross-reactivity concerns when working with CH25H antibody:

• Sequence Homology Analysis: Identify proteins with sequence similarity to CH25H that might cross-react with the antibody.

• Epitope Mapping: Determine the specific epitope recognized by the CH25H antibody to predict potential cross-reactivity.

• Preabsorption Controls: Perform parallel experiments with antibody preabsorbed with recombinant CH25H to confirm signal specificity.

• Immunoblotting Profile Analysis: Closely examine all bands detected by the antibody, not just those at the expected molecular weight of CH25H.

• Orthogonal Detection Methods: Confirm findings using alternative techniques such as mass spectrometry or PCR.

• Immunodepletion Studies: Sequentially immunodeplete samples with CH25H antibody and analyze remaining proteins for cross-reactivity.

These approaches collectively enhance confidence in the specificity of experimental results obtained using CH25H antibody (1G8).

What are effective strategies for optimizing immunoprecipitation protocols with CH25H antibody?

For successful immunoprecipitation of CH25H protein, consider these optimization strategies:

• Lysis Buffer Composition: Formulate buffers that maintain CH25H structure while effectively solubilizing membrane-associated proteins.

• Antibody-to-Protein Ratio: Titrate CH25H antibody concentrations to determine optimal binding without saturating the system.

• Incubation Conditions: Test various temperature and time combinations for antibody-antigen binding.

• Bead Selection: Compare protein A/G beads, magnetic beads, and direct-conjugated beads for optimal pull-down efficiency.

• Washing Stringency: Balance between removing non-specific interactions and maintaining specific antibody-CH25H complexes.

• Elution Methods: Evaluate various elution strategies including low pH, high salt, or competitive elution.

• Co-Immunoprecipitation Considerations: When studying CH25H interactions with other proteins, modify protocols to preserve protein-protein complexes.

Successful immunoprecipitation protocols should yield clean, specific isolation of CH25H for downstream applications such as interaction studies or functional assays.

How does CH25H expression correlate with various disease states?

Current research suggests connections between CH25H expression and several pathological conditions:

• Metabolic Disorders: Altered CH25H expression may contribute to dysregulated cholesterol metabolism in conditions like atherosclerosis and non-alcoholic fatty liver disease.

• Inflammatory Diseases: CH25H upregulation has been observed in response to inflammatory stimuli, suggesting roles in conditions like rheumatoid arthritis and inflammatory bowel disease.

• Viral Infections: 25-hydroxycholesterol produced by CH25H has demonstrated antiviral properties against certain enveloped viruses.

• Neurodegenerative Conditions: Emerging evidence suggests links between CH25H activity and neuroinflammatory processes in conditions like Alzheimer's disease.

When investigating these associations, researchers should employ CH25H antibody in combination with disease-specific markers to establish correlations and potential causative relationships between CH25H expression and pathological processes.

What are the latest methodological approaches for studying CH25H's role in cellular signaling pathways?

Cutting-edge approaches for investigating CH25H's signaling functions include:

• Proximity Labeling Techniques: BioID or APEX2 fusion proteins to identify proteins in close proximity to CH25H in living cells.

• Live-Cell Imaging: Fluorescently tagged CH25H to monitor real-time localization and trafficking in response to stimuli.

• CRISPR-Cas9 Gene Editing: Precise modification of CH25H or interacting proteins to assess functional relationships.

• Phosphoproteomics: Mass spectrometry-based approaches to identify signaling changes downstream of CH25H activation.

• Single-Cell Analysis: Examination of CH25H expression heterogeneity using flow cytometry or single-cell RNA sequencing.

• Oxysterol Sensors: Genetically encoded biosensors to track 25-hydroxycholesterol production and signaling in real-time.

These advanced methodologies complement traditional approaches using CH25H antibody (1G8) in fixed samples and can provide dynamic information about CH25H's roles in cellular signaling networks.

How can researchers effectively interpret contradictory data regarding CH25H function?

When faced with contradictory results in CH25H research, implement these analytical strategies:

• Contextual Analysis: Evaluate experimental conditions, cell types, and tissue sources across studies, as CH25H function may be context-dependent.

• Temporal Considerations: Assess whether contradictions might result from examining different time points in dynamic processes.

• Methodological Comparison: Scrutinize antibody validation, detection methods, and experimental protocols across contradictory studies.

• Isoform and Post-Translational Modification Analysis: Determine whether discrepancies might reflect different CH25H isoforms or modification states.

• Genetic Background Evaluation: Consider how genetic variations might influence CH25H function across experimental models.

• Hypothesis Reconciliation: Develop unified models that account for apparently contradictory observations, possibly suggesting multifunctional or context-specific roles for CH25H.

This systematic approach to data interpretation advances understanding of CH25H biology despite apparent contradictions in the published literature.

What emerging technologies might enhance CH25H antibody applications in research?

Several emerging technologies hold promise for expanding CH25H antibody utility:

• High-Throughput Antibody Validation: Advanced screening methods to more comprehensively validate antibody specificity across diverse conditions .

• Bispecific Antibody Development: Engineering antibodies that simultaneously target CH25H and interacting partners to study protein complexes .

• Recombinant Antibody Production: Synthetic gene technologies allowing precise antibody engineering for improved specificity and reduced batch variation .

• Complement Activation Assays: Integration of complement cascade measurements to evaluate immunological consequences of CH25H activity .

• Single-Domain Antibodies: Development of nanobodies or other minimal antibody formats for improved tissue penetration and intracellular targeting.

• Antibody-Drug Conjugates: Coupling CH25H antibodies with functional moieties for targeted modulation of cholesterol metabolism pathways.

These technological advances may significantly enhance researchers' ability to precisely study CH25H biology in increasingly complex experimental systems.

What are the most promising directions for translational research involving CH25H?

Translational research opportunities involving CH25H include:

• Metabolic Disease Therapeutics: Targeting CH25H activity to modulate cholesterol metabolism in hypercholesterolemia and related conditions.

• Anti-Inflammatory Applications: Exploring 25-hydroxycholesterol's immunomodulatory properties for inflammatory conditions.

• Antiviral Strategies: Developing approaches that leverage CH25H's role in producing antiviral oxysterols.

• Biomarker Development: Evaluating CH25H or 25-hydroxycholesterol as diagnostic or prognostic indicators for metabolic or inflammatory diseases.

• Drug Development: Screening compounds that modulate CH25H activity as potential therapeutic candidates.

Researchers pursuing these translational directions should consider combining CH25H antibody-based detection methods with functional assays that directly measure enzymatic activity and downstream effects.