Copper-metallothionein Antibody, Biotin conjugated

What is metallothionein and what is its relationship with copper?

Metallothioneins (MTs) are low-molecular-weight, cysteine-rich proteins that play crucial roles in metal ion homeostasis. These proteins are well-known for sensing and binding excess metals including Zn(II), Cd(II), and Cu(I) in cells, providing protection against metal toxicity. MTs function as chelators for harmful heavy metals and excessive essential metals, scavengers of radicals and reactive oxygen species, and regulators in cell proliferation processes. Their specific copper-binding capability makes MTs key proteins in copper homeostasis maintenance in various organisms .

What are the main characteristics of biotin-conjugated copper-metallothionein antibodies?

Biotin-conjugated copper-metallothionein antibodies consist of anti-metallothionein antibodies chemically linked to biotin molecules. These antibodies maintain their specificity for metallothionein while gaining the advantage of biotin's strong affinity for streptavidin/avidin systems. Key characteristics include:

• Available in both monoclonal and polyclonal formats depending on the research needs

• Can be produced from various host animals including mouse and rabbit

• Typically require protein G purification to ensure high specificity

• Maintain their MT recognition capabilities after biotin conjugation

• Most preparations contain stabilizers (e.g., glycerol) and bacteriostats (e.g., proclin) for extended shelf-life

What are the species-specific variants of metallothionein antibodies available for research?

Metallothionein antibodies show varying degrees of cross-reactivity across species. Available antibodies include:

This diversity allows researchers to select antibodies appropriate for their specific experimental system and target organism .

How can copper-metallothionein antibodies be effectively used in Western blotting protocols?

For optimal Western blotting results with biotin-conjugated copper-metallothionein antibodies:

• Use the recommended dilution ratio, typically 1:1000 for Western blotting applications

• Block membranes thoroughly to prevent non-specific binding

• Incubate with the biotin-conjugated primary antibody at appropriate temperature (usually room temperature for 1-2 hours or 4°C overnight)

• Detect using streptavidin-conjugated reporter systems (HRP, fluorescent tags)

• For Pseudomonas aeruginosa metallothionein, expect to detect a 36 kDa band

• Include positive controls using recombinant metallothionein proteins when possible

This approach leverages the sensitivity enhancement provided by the biotin-streptavidin detection system while maintaining specificity for metallothionein targets .

How can researchers use metallothionein antibodies to study copper-related disease mechanisms?

Metallothionein antibodies are valuable tools for investigating copper dysregulation in disease models:

• In neurodegenerative diseases like ALS, MT-I overexpression has been shown to normalize copper dyshomeostasis in the spinal cord of SOD1(G93A) mice

• MT-I overexpression significantly extends lifespan and slows disease progression in ALS models

• Use antibodies to monitor MT levels and localization in response to copper dyshomeostasis

• Study how MT overexpression attenuates pathological features including motor neuron death and glial cell activation

• Investigate how MT-I reduces SOD1 aggregates within glial cells and modulates caspase-3 activation

• Use MT antibodies in combination with copper chelators or supplementation to track cellular responses

These approaches can provide insights into the therapeutic potential of targeting copper regulation by MT-I in copper-related pathologies .

How can the MT1F promoter be utilized as a copper reporter system in research?

Based on screening of copper-responsive MTs in cancer cells, MT1F promoter has been established as an effective copper reporter system:

• Construct stable cell lines with MT1F promoter-driven EGFP (PMT1F-EGFP) as reporters

• This system specifically and stably reports intracellular Cu(I) changes across multiple cell lines (Panc-1, 8988T, 293T, HepG2, and normal hepatic cells)

• Use for real-time monitoring of copper fluctuations in living cells

• Apply to screen for compounds that affect cellular copper levels (e.g., MEK inhibitors like U0126 and Astragaloside IV have been found to significantly increase intracellular copper ions)

• Employ to study copper-related cellular physiology and pathology

This approach offers advantages over traditional methods by providing real-time, in vivo monitoring of copper status in intact cellular systems .

What are the critical factors affecting the biotin switch assay when working with copper-metallothionein?

The biotin switch assay, often used to study protein modifications including those of metallothioneins, is significantly influenced by copper levels:

• "Contaminating" copper is required for the ascorbate-dependent degradation of S-nitrosothiol, a critical step in the assay

• Removal of copper from buffers by chelators like DTPA preserves approximately 90% of S-nitrosothiol and decreases biotin labeling

• Addition of copper and ascorbate completely eliminates S-nitrosothiol and increases specific biotin labeling

• Reduced copper (Cu+) is specifically required for the degradation of RSNO to free thiol for subsequent biotinylation

• For consistent results, prepare a saturated CuCl solution (approximately 100 μM) by vortexing in HPLC grade water, centrifuging, and adding the supernatant to the biotinylation reaction

• Variable results between laboratories likely stem from differences in buffer composition and inherent copper content

Understanding these copper-dependent aspects is essential for reproducible results when working with metallothionein modifications and biotin labeling strategies .

What storage and handling recommendations optimize the performance of biotin-conjugated copper-metallothionein antibodies?

To maintain antibody functionality and extend shelf-life:

• Store unopened antibodies at -20°C as recommended by manufacturers

• Once thawed, store at 4°C to avoid repeated freeze-thaw cycles

• For long-term storage, aliquot the antibody solution to minimize freeze-thaw cycles

• Buffer formulations typically contain 50% glycerol and 0.03% Proclin-300 as stabilizers

• Verify antibody activity periodically using positive controls

• Determine optimal dilutions empirically for each application, starting with manufacturer recommendations

• For Biotin-conjugated MT antibodies, maintain refrigerated temperature during handling to preserve biotin-antibody linkage

Following these recommendations maximizes antibody performance and extends usable lifespan .

How can researchers distinguish between different metallothionein isoforms using antibodies?

Distinguishing between metallothionein isoforms requires careful antibody selection and experimental design:

• Select antibodies raised against specific MT isoforms (e.g., MT1A, MT1E, MT1F)

• Verify antibody specificity using recombinant MT isoforms as positive controls

• For closely related isoforms, consider using RNA-level detection methods (qPCR) alongside protein detection

• Validate antibody specificity through immunoprecipitation followed by mass spectrometry

• MT isoform expression varies by tissue and in response to different stimuli - incorporate appropriate tissue controls

• When using MT1F as a copper reporter, confirm that the response is specific to copper and not general cellular stress

• For antibodies like monoclonal 1A12 that recognize multiple MT isoforms, use complementary approaches to identify specific isoforms

This multi-faceted approach enables more precise characterization of specific metallothionein isoforms in complex biological samples .

How do copper chaperones interact with metallothioneins in cellular copper trafficking networks?

Copper chaperones work in concert with metallothioneins to control cellular copper distribution:

• Copper chaperone CCS interacts with MEK1 at an apparent dissociation constant (Kd) of 2.09 ± 0.48 μM

• The association between CCS and target proteins appears transient, as demonstrated by electrophoretic mobility shift assays and size-exclusion chromatography

• BirA proximity-dependent biotin identification (BioID) techniques capture these weak/transient interactions in living cells

• Streptavidin recovery of biotinylated MEK1 is significantly reduced by copper chelators like BCS

• CCS-facilitated copper delivery to proteins like MEK1/2 modulates kinase activity and cellular signaling

• Metallothioneins likely serve as copper reservoirs that interface with the chaperone network

• This copper trafficking network represents a potential target for therapeutic intervention in diseases with copper dyshomeostasis

Understanding these interactions provides insights into how cells precisely distribute copper to various proteins and compartments .

What molecular mechanisms underlie the protective effects of metallothionein overexpression in neurodegenerative disease models?

Metallothionein overexpression provides neuroprotection through multiple mechanisms:

• In SOD1(G93A) ALS mouse models, MT-I overexpression normalizes copper dyshomeostasis in the spinal cord without affecting SOD1 enzymatic activity

• MT-I attenuates multiple pathological features including motor neuron death, degeneration of ventral root axons, skeletal muscle atrophy, and glial cell activation

• Overexpression of MT-I decreases SOD1 aggregates within glial cells

• MT-I reduces the number of spheroid-shaped astrocytes cleaved by active caspase-3, suggesting anti-apoptotic effects

• MT-I acts as a copper-regulating protein that can chelate excess copper and provide copper when needed

• The protective effects translate to significant life extension and delayed disease progression in ALS models

These findings suggest therapeutic potential for approaches that enhance MT-I expression or mimic its copper-regulating functions in neurodegenerative diseases .

How can researchers effectively combine copper chelators with metallothionein antibodies to study protein-metal interactions?

To investigate protein-metal interactions using both chelators and antibodies:

• Design experiments with sequential or simultaneous application of copper chelators (e.g., BCS, DTPA) and MT antibodies

• Use cell-permeable versus cell-impermeable chelators to distinguish between intracellular and extracellular copper pools

• In biotin switch assays, control copper levels precisely, as chelators like DTPA preserve S-nitrosothiols while addition of copper enhances biotin labeling

• When studying CCS interactions, note that streptavidin recovery of biotinylated MEK1 is significantly reduced by copper chelators

• For MT1F-based copper reporters, chelators can validate the specificity of the copper response

• Create dose-response curves with increasing chelator concentrations to determine the relationship between copper availability and MT binding/function

• Use complementary techniques like atomic absorption spectroscopy to quantify metal content

This combined approach provides deeper insights into the dynamic relationship between copper availability and metallothionein function .

What are the current challenges and future directions in copper-metallothionein antibody development for advanced research applications?

Current challenges and emerging opportunities include:

• Developing antibodies with greater isoform specificity to distinguish between MT-1 subtypes (MT1A, MT1E, MT1F, MT1X, etc.)

• Creating antibodies that selectively recognize metal-bound versus metal-free metallothioneins

• Designing antibodies that can distinguish between different metal ions (Zn vs. Cu vs. Cd) bound to metallothionein

• Incorporating novel conjugates beyond biotin (e.g., quantum dots, photo-switchable fluorophores) for super-resolution imaging

• Developing antibodies suitable for in vivo imaging of metallothionein dynamics

• Addressing cross-reactivity issues between closely related metallothionein family members

• Creating antibody tools to study the interfaces between metallothioneins and copper chaperones

Advances in these areas would significantly enhance our ability to study the complex roles of metallothioneins in copper homeostasis and disease processes .

How can copper-metallothionein antibodies contribute to understanding copper dysregulation in amyotrophic lateral sclerosis?

Copper-metallothionein antibodies provide valuable insights into ALS pathophysiology:

• Track MT-I expression levels in SOD1 mutant mouse models using immunohistochemistry and Western blotting

• Examine the relationship between MT-I levels and disease progression in different CNS regions

• Investigate how MT-I overexpression normalizes copper dyshomeostasis without affecting SOD1 enzymatic activity

• Use antibodies to assess the presence of MT-I in motor neurons versus glial cells during disease progression

• Study how MT-I overexpression reduces SOD1 aggregates and decreases caspase-3 activation in astrocytes

• Compare MT-I expression patterns between familial and sporadic ALS cases

• Evaluate MT-I as a potential biomarker for disease progression or treatment response

These approaches contribute to understanding copper regulation as a therapeutic target in ALS and potentially other neurodegenerative diseases .

What experimental protocols enable accurate quantification of metallothionein levels in biological samples?

For reliable quantification of metallothioneins:

• ELISA using biotinylated anti-metallothionein antibodies provides sensitive detection with a working dilution of approximately 0.01μg/mL

• For Western blotting, use dilutions around 1:1000 with appropriate protein loading controls

• Include recombinant metallothionein standards to generate calibration curves

• For complex samples, consider immunoprecipitation followed by Western blotting

• When working with S-nitrosylated metallothioneins, account for copper dependency in biotin switch assays

• For cells expressing GFP under MT1F promoter control, quantify fluorescence intensity as a surrogate for MT expression

• Consider complementary mRNA quantification using qPCR for MT isoforms with high sequence similarity

• For tissue samples, immunohistochemistry with biotin-conjugated antibodies allows spatial localization and semi-quantitative analysis

These protocols ensure accurate measurement of metallothionein levels across different experimental systems and biological contexts .

How might advances in antibody engineering enhance the specificity and utility of copper-metallothionein antibodies?

Emerging antibody technologies offer new possibilities:

• Single-domain antibodies (nanobodies) may provide superior access to cryptic metallothionein epitopes

• Antibody fragments with enhanced tissue penetration could improve in vivo studies

• Bispecific antibodies targeting metallothionein and copper chaperones might reveal interaction dynamics

• CRISPR-guided epitope tagging combined with anti-tag antibodies could enhance specificity for particular MT isoforms

• Antibody phage display technologies may yield highly specific antibodies against conformational epitopes present only in metal-bound MTs

• Intrabodies expressed within specific cellular compartments could track MT localization in real-time

• Proximity-labeling antibodies could identify novel MT-interacting proteins in the copper trafficking network

These innovations promise to overcome current limitations in studying metallothionein biology and copper homeostasis .

What opportunities exist for developing metallothionein-targeted therapeutics for copper-related disorders?

Metallothionein-based therapeutic strategies show promise:

• MT-I overexpression significantly extends lifespan in ALS mouse models, suggesting gene therapy approaches

• Small molecules that induce endogenous MT expression could provide therapeutic benefits

• Peptide mimetics of MT metal-binding domains might serve as targeted copper chelators

• Antibody-drug conjugates could deliver copper-modulating agents to specific tissues

• Biomarkers based on MT isoform expression patterns might enable personalized treatment approaches

• Combination therapies targeting both metallothioneins and copper chaperones could provide synergistic effects

• Novel copper-binding compounds inspired by MT structure could offer improved pharmacokinetic properties