Copper-metallothionein Antibody, HRP conjugated

What is Copper-metallothionein and why is it important in biological systems?

Metallothioneins (MTs) are low-molecular-weight, cysteine-rich proteins that play critical roles in metal homeostasis. Copper-metallothionein specifically refers to MT molecules that have bound copper ions. These proteins are essential for:

• Protection against metal toxicity through tight chelation of copper ions

• Regulation of intracellular copper distribution

• Modulation of copper-dependent cellular processes

• Protection against oxidative stress

Research has shown that metallothioneins contain specific metal-binding domains that can accommodate multiple copper atoms per polypeptide chain. For instance, MT3 contains three zinc and three copper atoms per polypeptide chain with negligible amounts of cadmium . These proteins are transcriptionally regulated by both heavy metals and glucocorticoids, making them important markers for metal exposure and stress responses .

What applications are suitable for Copper-metallothionein Antibody, HRP conjugated?

Copper-metallothionein Antibody with HRP conjugation is primarily used in the following research applications:

The HRP (horseradish peroxidase) conjugation enables direct enzymatic detection without the need for secondary antibodies, making it particularly valuable for ELISA-based experiments where streamlined workflows are desired .

What species reactivity can be expected with commercial Copper-metallothionein Antibodies?

Available commercial antibodies show distinct species reactivity profiles:

When selecting an antibody for your research, it's crucial to choose one validated for your species of interest. The CUP1-1 antibody specifically targets yeast copper metallothionein and has been optimized for studies in Saccharomyces cerevisiae .

How should I optimize ELISA protocols when using Copper-metallothionein Antibody, HRP conjugated?

Optimizing ELISA protocols with HRP-conjugated Copper-metallothionein Antibody requires attention to several key parameters:

• Antibody Dilution: Start with manufacturer-recommended dilutions, typically 1:500-1:1000 for ELISA applications . Perform a titration experiment to determine the optimal antibody concentration for your specific sample type.

• Blocking Solution: Use a 1-5% BSA solution in PBS to minimize background. Avoid milk-based blockers which may contain endogenous phosphatases that could interfere with HRP activity.

• Incubation Conditions:

  • Primary antibody (HRP-conjugated): 1-2 hours at room temperature or overnight at 4°C

  • Substrate development: 5-30 minutes at room temperature (monitor color development)

• Substrate Selection: TMB (3,3',5,5'-Tetramethylbenzidine) is recommended for HRP detection due to its sensitivity and low background.

• Controls: Always include:

  • Positive control (known copper-metallothionein sample)

  • Negative control (sample without target protein)

  • Background control (no primary antibody)

The optimal working dilution should be determined by the investigator for each specific application and sample type .

What are the best sample preparation methods for detecting Copper-metallothionein in different tissue types?

Sample preparation varies significantly based on tissue type and experimental goals:

For Cell Lysates (Western Blot):

• Harvest cells and wash twice with ice-cold PBS

• Lyse cells using a buffer containing:

  • 50 mM Tris-HCl, pH 7.5

  • 150 mM NaCl

  • 1% NP-40 or Triton X-100

  • Protease inhibitor cocktail

• Include metal chelators (e.g., EDTA) cautiously as they may disrupt copper-metallothionein interactions

• Centrifuge at 14,000g for 15 minutes at 4°C and collect supernatant

• Determine protein concentration before loading

For Tissue Sections (Immunohistochemistry):

• Fix tissue with formaldehyde and embed in paraffin

• Perform antigen retrieval by heat mediation (crucial for metallothionein detection)

• Block with protein block for 10 minutes at room temperature

• Apply antibody at appropriate dilution (typically 1:150 for MT antibodies)

• Incubate for 30 minutes at room temperature followed by secondary detection

For Yeast Samples (Specific to CUP1-1):

• Culture Saccharomyces cerevisiae under appropriate copper conditions

• Perform spheroplasting to improve protein extraction efficiency

• Use glass bead lysis in buffer containing DTT to preserve protein structure

• Avoid excessive heat during preparation which may denature the target

Proper sample preparation is critical as metallothioneins are relatively small proteins that can be easily degraded during processing .

How can I validate the specificity of my Copper-metallothionein Antibody results?

Validating antibody specificity is crucial for reliable metallothionein research. Implement these approaches:

• Peptide Competition Assay: Pre-incubate the antibody with excess synthetic Copper-metallothionein peptide before application. Specific signals should be significantly reduced or eliminated.

• Knockout/Knockdown Controls: Use samples from metallothionein knockout models or cells with siRNA-mediated knockdown as negative controls.

• Parallel Antibody Validation: Compare results using multiple antibodies targeting different epitopes of the same protein.

• Recombinant Protein Standards: Include purified recombinant Copper-metallothionein protein as a positive control to verify detection at the correct molecular weight.

• Induction Experiments: Treat cells with copper or other heavy metals known to induce metallothionein expression and confirm increased signal as expected.

• Mass Spectrometry Correlation: When possible, correlate antibody detection with mass spectrometry analysis of the same samples for orthogonal validation.

Remember that metallothioneins are small proteins (~6-7 kDa) that may migrate anomalously on some gel systems, making molecular weight verification particularly important .

How do different metal ions affect the detection of metallothioneins by antibodies?

The metal-binding status of metallothioneins significantly impacts their detection by antibodies through conformational changes:

• Conformational Effects: Metallothioneins undergo substantial conformational changes when binding different metal ions. These changes can expose or mask epitopes recognized by antibodies.

• Metal-Specific Considerations:

  • Copper-bound MT: Often displays a more compact structure that may reduce antibody accessibility to some epitopes

  • Zinc-bound MT: Generally maintains a more open conformation

  • Cadmium-bound MT: May induce distinct conformational changes affecting antibody recognition

• Mixed Metal Populations: In biological samples, metallothioneins typically contain mixed metal populations, which creates heterogeneous protein conformations and variable antibody recognition.

• Technical Recommendations:

  • When analyzing copper-specific metallothionein binding, consider treating samples with controlled copper concentrations

  • Include metal chelation controls (EDTA/EGTA treatment) to assess apo-metallothionein detection

  • For quantitative studies, standardize the metal content of reference materials

Research has shown that the three-dimensional structure of metallothionein varies significantly depending on bound metals, which can affect antibody recognition efficiency by up to 40-60% depending on the specific epitope targeted .

What are the technical challenges in distinguishing between different metallothionein isoforms?

Distinguishing between metallothionein isoforms presents several technical challenges:

• High Sequence Homology: MT isoforms share significant sequence similarity, with human MT1 and MT2 sharing approximately 70% sequence identity, making specific antibody development challenging.

• Small Size: MTs are small proteins (~6-7 kDa) with limited epitope diversity, restricting options for isoform-specific antibody generation.

• Post-translational Modifications: Metal binding and oxidation states create heterogeneity within each isoform population.

• Isoform-Specific Strategies:

  • Use recombinant expression systems to generate isoform-specific standards

  • Employ epitope mapping to identify unique regions for antibody development

  • Consider complementary techniques like mass spectrometry for isoform differentiation

• Validation Approaches:

  • Overexpression systems with tagged isoforms

  • Isoform-specific knockdown

  • Correlation with mRNA expression of specific isoforms

When studying specific isoforms like MT3, carefully validate antibody specificity against other MT family members. Research has shown that antibodies targeting the N-terminal region tend to provide better isoform specificity, while those targeting the metal-binding domains often show cross-reactivity .

How can I troubleshoot non-specific binding when using Copper-metallothionein Antibody, HRP conjugated?

Non-specific binding is a common challenge with metallothionein antibodies. Address it systematically:

• Common Sources of Non-specific Binding:

  • Excessive antibody concentration

  • Insufficient blocking

  • Problematic sample preparation

  • Cross-reactivity with related metallothionein isoforms

• Optimization Strategies:

  • Antibody Titration: Test a range of dilutions (1:1000, 1:2000, 1:5000, 1:10000) to identify optimal signal-to-noise ratio

  • Blocking Enhancement: Increase blocking time (2-4 hours) or concentration (3-5% BSA)

  • Buffer Optimization: Add 0.1-0.5% Tween-20 to wash buffers to reduce hydrophobic interactions

  • Sample Pre-treatment: Pre-absorb samples with irrelevant proteins

• HRP-Specific Considerations:

  • Endogenous peroxidase activity can be quenched with 0.3% H₂O₂ treatment

  • Use peroxidase-free blocking reagents

  • Consider shorter substrate development times to minimize background

• Validation Controls:

  • Include isotype control antibodies at the same concentration

  • Perform peptide competition assays

  • Include samples lacking the target protein

Remember that metallothioneins contain high cysteine content which may contribute to non-specific interactions through disulfide bridge formation. Including reducing agents in sample buffers can help mitigate this issue .

How can Copper-metallothionein Antibody be used to study metal-induced stress responses?

Copper-metallothionein antibodies provide valuable tools for investigating metal-induced stress responses:

• Experimental Design Approaches:

  • Dose-Response Studies: Treat cells with increasing concentrations of copper or other metals and quantify metallothionein induction using antibody-based detection methods.

  • Time-Course Analysis: Monitor the kinetics of metallothionein expression following metal exposure to understand temporal aspects of the stress response.

  • Co-localization Studies: Combine metallothionein antibodies with organelle markers to track intracellular metallothionein distribution during stress.

• Methodological Applications:

  • Quantitative Western Blotting: Measure total metallothionein protein levels

  • Immunocytochemistry: Visualize subcellular localization and trafficking

  • Flow Cytometry: Analyze metallothionein expression at the single-cell level

  • ELISA: Quantify metallothionein in biological fluids or cell culture supernatants

• Biomarker Potential:
  Research has demonstrated that metallothionein levels serve as sensitive biomarkers for metal exposure and oxidative stress. Elevated antibodies to metallothionein have been found in patients with metal allergies, with significantly higher positive frequencies (51.3%) compared to healthy controls (3.8%) .

• Comparative Studies:
  Using antibodies against different metallothionein isoforms allows researchers to distinguish isoform-specific responses to different stressors, as MT-1/MT-2 and MT-3 show distinct expression patterns and regulation mechanisms .

What role do metallothioneins play in copper homeostasis and how can antibodies help elucidate these mechanisms?

Metallothioneins play crucial roles in copper homeostasis that can be investigated using antibody-based approaches:

• Key Homeostatic Functions:

  • Buffering cytosolic copper concentrations

  • Detoxification of excess copper

  • Storage of copper for later utilization

  • Protection against copper-induced oxidative damage

• Molecular Interactions:
  Recent research has revealed that metallothioneins regulate ATP7A trafficking in a copper-dependent manner. Studies show that a lack of metallothioneins results in trafficking of ATP7A from the trans-Golgi complex, suggesting that metallothioneins regulate the delivery of copper to ATP7A .

• Antibody-Based Investigation Approaches:

  • Co-immunoprecipitation: Identify metallothionein-interacting proteins involved in copper transport and regulation

  • Proximity Ligation Assays: Visualize in situ interactions between metallothioneins and copper transport proteins

  • ChIP-seq: Analyze transcription factor binding to metallothionein promoters under copper stress

• Functional Studies:

  • Track metallothionein expression and localization during copper deficiency and excess

  • Compare wild-type and metallothionein-deficient models using immunodetection

  • Correlate metallothionein levels with copper-dependent enzyme activities

Research has demonstrated that metallothioneins are critical for copper tolerance in the absence of ATP7A, with combined deficiency of both systems resulting in extreme copper sensitivity . This synthetic lethal phenotype highlights the complementary roles of these proteins in maintaining copper homeostasis.

How can metallothionein antibodies contribute to understanding neurodegenerative diseases?

Metallothionein antibodies provide valuable tools for investigating the role of these proteins in neurodegenerative processes:

• MT3-Specific Neurological Functions:
  Metallothionein-3 (MT3), originally identified as Growth Inhibitory Factor (GIF), plays unique roles in the central nervous system:

  • Inhibits survival and neurite formation of cortical neurons in vitro

  • Regulates copper homeostasis in the brain

  • Protects against metal-induced neurotoxicity

  • May be dysregulated in Alzheimer's disease and other neurodegenerative conditions

• Research Applications:

  • Comparative Expression Analysis: Quantify MT3 levels in healthy vs. diseased brain tissue using immunohistochemistry

  • Cellular Studies: Investigate MT3 localization in different neural cell populations

  • Animal Models: Track metallothionein expression changes in neurodegenerative disease models

  • Biomarker Development: Assess metallothionein levels in cerebrospinal fluid as potential diagnostic markers

• Technical Considerations:

  • Use antibodies specifically validated for neural tissues

  • Consider brain region-specific differences in metallothionein expression

  • Account for age-related changes in metallothionein levels

• Therapeutic Implications:
  Understanding metallothionein regulation in neurodegenerative contexts may lead to novel therapeutic approaches. Recent research suggests that MT3 as a built-in adjuvant has potential for vaccine development, which could have implications for treating certain neurological conditions .

For neurological studies, metallothionein-3 antibodies with validated reactivity in human and mouse brain tissues are particularly valuable, as they allow translation between animal models and clinical samples .

How can metallothionein antibodies be utilized in immunomodulatory research?

Recent discoveries reveal exciting potential for metallothioneins in immunomodulation that can be investigated using antibody-based approaches:

• Novel Adjuvant Properties:
  Groundbreaking research has demonstrated that metallothionein-3 (MT3) functions as a built-in adjuvant that can help protein antigens induce rapid, effective, and durable antigen-specific immune responses:

  • MT3 fusion proteins increased antigen-specific antibody responses by 100-1000 fold within seven days after primary immunization

  • MT3 stimulated earlier (4 days post-injection) and stronger (10-100 fold) antibody responses compared to commercial adjuvants

  • MT3 allowed significant antigen dose sparing while maintaining robust immune responses

• Research Applications:

  • Comparative Adjuvant Studies: Compare MT3 with traditional adjuvants using antibody-based detection of immune responses

  • Mechanistic Investigations: Explore how MT3 activates dendritic cells and promotes germinal center formation

  • Fusion Protein Development: Design and evaluate novel MT3-antigen fusion constructs

• Technical Approaches:

  • Use anti-metallothionein antibodies to track MT3 localization in immune tissues

  • Develop ELISAs to monitor antibody responses to MT3-fusion antigens

  • Apply immunoprecipitation to identify MT3-interacting immune factors

This emerging field represents a paradigm shift in understanding metallothioneins beyond their traditional metal-binding roles, with potential applications in vaccine development against global pandemics .

What are the latest findings on metallothionein antibodies as biomarkers for metal-related health conditions?

Metallothionein antibodies show promising potential as biomarkers for various metal-related health conditions:

• Metal Allergy Biomarkers:
  Research has demonstrated that antibodies to metallothionein exist in human serum and are significantly elevated in metal allergy cases:

  • High positive frequency of antibody to MT (51.3%) in patients with metal allergy compared to healthy controls (3.8%)

  • Strong positive correlation between antibodies to MT and heat shock protein 70 (hsp70) specifically in metal allergy patients

  • Positive rates for both antibodies were significantly higher in metal allergy patients than in atopic dermatitis patients without metal allergy

• Potential Clinical Applications:

  • Diagnostic Assays: Development of serological tests for metal sensitivity using anti-metallothionein antibodies

  • Exposure Assessment: Correlation of metallothionein antibody levels with environmental or occupational metal exposure

  • Treatment Monitoring: Tracking metallothionein antibody levels during chelation therapy

• Research Methodologies:

  • Multiplex Assays: Simultaneous detection of antibodies against multiple metallothionein isoforms

  • Epitope Mapping: Identification of metal-specific epitopes recognized by autoantibodies

  • Longitudinal Studies: Monitoring antibody levels over time to assess chronicity

• Mechanistic Insights:
  The presence of anti-metallothionein antibodies suggests potential autoimmune mechanisms in metal toxicity, where extensive cell destruction due to chronic metal exposure may lead to release of metallothioneins and subsequent antibody production .

How can advanced microscopy techniques enhance metallothionein research using specialized antibodies?

Advanced microscopy techniques paired with metallothionein antibodies enable sophisticated analyses of protein localization and dynamics:

• Super-Resolution Microscopy Approaches:

  • STED (Stimulated Emission Depletion): Resolve metallothionein distribution at sub-diffraction resolution (20-50 nm)

  • PALM/STORM: Track single metallothionein molecules with 10-20 nm precision

  • SIM (Structured Illumination Microscopy): Achieve 2x conventional resolution improvement for metallothionein visualization

• Live-Cell Imaging Applications:

  • FRAP (Fluorescence Recovery After Photobleaching): Measure metallothionein mobility in different cellular compartments

  • FRET (Förster Resonance Energy Transfer): Detect interactions between metallothioneins and copper transport proteins

  • Optogenetic Approaches: Combine light-controlled systems with metallothionein visualization

• Correlative Microscopy Integration:

  • CLEM (Correlative Light and Electron Microscopy): Combine immunofluorescence with ultrastructural analysis

  • LA-ICP-MS Imaging: Correlate metallothionein localization with metal distribution

  • Raman Microscopy: Analyze metal-protein interactions with spectroscopic information

• Technical Considerations:

  • Use directly labeled primary antibodies for improved resolution

  • Consider small epitope tags for live-cell applications

  • Validate colocalization with appropriate controls and quantitative analysis

For metallothionein trafficking studies, fluorescently labeled antibodies or antibody fragments have been used to track the protein's movement in response to changing copper levels. Research has shown that metallothioneins influence the trafficking of ATP7A from the trans-Golgi complex in a copper-dependent manner, demonstrating their role in copper homeostasis beyond simple metal sequestration .

What are the prospects for developing isoform-specific metallothionein antibodies with enhanced specificity?

The development of highly specific metallothionein isoform antibodies represents an important frontier:

• Current Challenges:

  • High sequence homology between metallothionein isoforms (70-90% for MT1/MT2)

  • Small protein size limiting epitope diversity

  • Conformational changes due to metal binding affecting epitope accessibility

• Innovative Approaches:

  • Recombinant Antibody Engineering: Use phage display to select antibodies with enhanced isoform specificity

  • Unique Epitope Targeting: Focus on N-terminal regions where isoforms show greater sequence divergence

  • Conformation-Specific Antibodies: Develop antibodies that recognize specific metal-loaded states

  • Single-Domain Antibodies: Explore nanobodies for accessing restricted epitopes

• Validation Strategies:

  • Comprehensive cross-reactivity testing against all metallothionein isoforms

  • Knockout/knockdown validation in multiple cell types

  • Mass spectrometry confirmation of isoform-specific detection

• Potential Applications:

  • Precise quantification of isoform-specific expression patterns

  • Investigation of isoform-specific subcellular localization

  • Identification of unique binding partners for different isoforms

The development of truly isoform-specific antibodies would enable researchers to distinguish the diverse and sometimes opposing functions of different metallothionein family members, particularly in complex systems like the nervous system where multiple isoforms are co-expressed .

How might metallothionein antibodies contribute to understanding the role of copper dyshomeostasis in disease?

Metallothionein antibodies provide valuable tools for investigating copper dyshomeostasis in various diseases:

• Disease Contexts for Investigation:

  • Neurodegenerative Disorders: Alzheimer's, Parkinson's, and ALS involve copper misregulation

  • Wilson's Disease: Genetic disorder of copper accumulation

  • Menkes Disease: Genetic disorder of copper deficiency

  • Cancer: Altered copper metabolism in tumor microenvironments

  • Inflammatory Conditions: Copper-mediated oxidative stress in chronic inflammation

• Research Applications:

  • Tissue Distribution Analysis: Map metallothionein expression changes in diseased vs. healthy tissues

  • Cellular Response Studies: Investigate how cells adapt metallothionein expression to copper stress

  • Genetic Model Systems: Compare metallothionein responses in disease-relevant genetic backgrounds

• Integrated Approaches:

  • Combine metallothionein detection with copper transport protein analysis (ATP7A, ATP7B, CTR1)

  • Correlate metallothionein levels with biomarkers of oxidative stress

  • Integrate metallothionein data with copper-dependent enzyme activities

• Therapeutic Implications:
  Recent research has revealed that metallothioneins regulate ATP7A trafficking and are critical for copper tolerance in the absence of ATP7A. The synthetic lethal phenotype observed when both systems are compromised highlights potential therapeutic targets for conditions involving copper dysregulation .

Understanding how metallothioneins function in copper homeostasis may lead to novel therapeutic strategies for diseases characterized by copper dyshomeostasis, potentially through targeted modulation of metallothionein expression or function.

What potential exists for metallothionein-based therapies and how can antibodies facilitate their development?

Metallothionein-based therapeutic strategies represent an emerging area with significant potential:

• Therapeutic Applications:

  • Adjuvant Development: MT3 fusion proteins for enhanced vaccine responses

  • Metal Chelation Therapy: Targeted metallothionein induction for metal detoxification

  • Neuroprotective Strategies: Metallothionein administration for neurodegenerative conditions

  • Anti-inflammatory Approaches: Leveraging metallothionein's antioxidant properties

• Antibody Contributions to Development:

  • Pharmacokinetic Studies: Track recombinant metallothionein distribution and half-life

  • Target Engagement: Confirm binding to intended cellular targets

  • Biomarker Development: Monitor endogenous metallothionein responses to therapy

  • Safety Assessment: Evaluate potential immunogenicity of metallothionein-based therapeutics

• Innovative Therapeutic Modalities:

  • MT-Fusion Proteins: Metallothionein fused to targeting moieties for specific delivery

  • MT-Mimetic Peptides: Synthetic peptides mimicking metallothionein functions

  • Gene Therapy: Targeted metallothionein expression in affected tissues

  • Small Molecule Inducers: Compounds that upregulate endogenous metallothionein expression

• Promising Research Findings:
  Research has demonstrated that MT3 fusion proteins can enhance antigen-specific immune responses by 100-1000 fold compared to antigen alone, with effective dose sparing. This adjuvant effect works specifically when MT3 is fused to the antigen and provides earlier, stronger responses than commercial adjuvants, suggesting significant potential for vaccine development against global pandemics .

The development of metallothionein-based therapeutics will require sophisticated antibody tools for characterization, optimization, and monitoring of these novel treatment approaches.

What are the recommended controls when working with Copper-metallothionein Antibody, HRP conjugated?

Implementing appropriate controls is essential for reliable results with Copper-metallothionein Antibody, HRP conjugated:

• Positive Controls:

  • Recombinant copper-metallothionein protein

  • Cell lines or tissues known to express high levels of metallothionein

  • Samples from metal-treated cells with induced metallothionein expression

• Negative Controls:

  • Isotype control antibody at matching concentration

  • Metallothionein knockout/knockdown samples

  • Secondary antibody-only controls (for non-conjugated primary antibodies)

• Technical Controls:

  • Endogenous peroxidase quenching controls for tissue sections

  • Signal specificity verification via peptide competition

  • Dilution series to confirm signal proportionality to target concentration

• Sample Processing Controls:

  • Parallel processing of all experimental conditions

  • Internal loading controls for western blots (β-actin, GAPDH)

  • Time-course controls for signal development

For optimal results with HRP-conjugated antibodies, manufacturer recommendations suggest including substrate-only wells to assess potential background from the detection system itself .

What are the key differences between various metallothionein antibodies available for research?

Various metallothionein antibodies offer distinct characteristics important for experimental design:

When selecting an antibody:

• Consider the specific metallothionein isoform relevant to your study

• Verify species reactivity with your experimental model

• Ensure the antibody is validated for your application

• Check if the conjugation status matches your detection system

Antibodies targeting different regions of metallothioneins may show varying sensitivity to metal-binding status, with some epitopes becoming masked when metals are bound .

What resources are available for researchers studying copper metabolism using metallothionein antibodies?

Researchers investigating copper metabolism with metallothionein antibodies can access various resources:

• Research Tools:

  • Antibodies: Commercially available metallothionein antibodies from vendors like Abcam, Cusabio, and Bioss

  • Recombinant Proteins: Purified metallothioneins for standards and controls

  • Cell Lines: Metallothionein-knockout and overexpression systems

  • Animal Models: MT-null mice for in vivo studies

• Technical Resources:

  • Protocols: Validated methodologies for metallothionein detection in various sample types

  • Databases: Metal-binding protein databases and metallothionein sequence repositories

  • Software: Image analysis tools for quantifying immunolabeling

• Scientific Literature:

  • Key studies on metallothionein regulation of ATP7A trafficking

  • Research on metallothionein antibodies as biomarkers for metal allergies

  • Investigations of metallothionein-3 as a built-in adjuvant

• Collaborative Networks:

  • International metallothionein research consortia

  • Specialized conferences on metal biology

  • Online research communities and forums