Copper-metallothionein Antibody, FITC conjugated

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

Copper-metallothionein (Cu-MT) is a low molecular weight, cysteine-rich protein that plays crucial roles in cellular copper homeostasis. Metallothioneins increase resistance to high copper levels by sequestering excess copper in a solvent-shielded core. Cu-MT functions primarily in:

• Safe intracellular copper storage

• Protection against copper toxicity and oxidative stress

• Possible delivery of copper ions to cuproenzymes

Research shows that metallothioneins are required for a cell's ability to accumulate copper when extracellular copper reaches physiological levels (10 μM) . In pathogenic organisms like Mycobacterium tuberculosis, copper-binding metallothioneins (such as MymT) can bind up to 6 Cu(I) ions and partially protect against copper toxicity .

What is the structure and specificity of FITC-conjugated Copper-metallothionein antibody?

The FITC-conjugated Copper-metallothionein antibody is a rabbit polyclonal antibody that specifically targets Copper-metallothionein. Key characteristics include:

The antibody shows primary reactivity with Roman snail (Helix pomatia) Cu-MT, though reactivity with other species should be tested empirically .

How can FITC-conjugated Copper-metallothionein antibody be used in immunofluorescence studies?

For immunofluorescence detection of Copper-metallothionein, the following protocol can be employed:

• Fix cells with 4% paraformaldehyde in PBS for 10 minutes at 4°C

• Wash cells with PBS buffer

• Permeabilize cells with 0.1% saponin in PBS for 20 minutes

• Incubate with FITC-conjugated Cu-MT antibody at the optimal dilution (typically 1-5 μg/ml) for 60 minutes in the dark

• Wash extensively with 0.1% saponin in PBS

• Mount and examine using a fluorescence microscope with appropriate filters for FITC detection (excitation ~495 nm, emission ~519 nm)

This direct detection method eliminates the need for secondary antibodies, reducing background and cross-reactivity issues that may occur in two-step detection methods .

What are the optimal conditions for using Cu-MT FITC antibody in flow cytometry?

For flow cytometric analysis of Copper-metallothionein expression:

• Harvest cells (1×10^6 cells/sample) and wash with cold PBS containing 1% BSA

• Fix cells with 2% paraformaldehyde for 15 minutes at room temperature

• Permeabilize with 0.1% saponin or 0.1% Triton X-100 in PBS for 10 minutes

• Incubate with FITC-conjugated Cu-MT antibody (1-5 μg/ml) for 30-45 minutes at room temperature in the dark

• Wash twice with permeabilization buffer

• Resuspend in PBS containing 1% BSA

• Analyze using a flow cytometer with 488 nm excitation and detection in the FL1 channel

When analyzing results, establish appropriate gating strategies based on negative controls to account for autofluorescence and non-specific binding .

How does sample preparation affect Cu-MT antibody detection efficiency?

Sample preparation significantly impacts detection efficiency of Copper-metallothionein:

For optimal detection:

• Perform fixation immediately after treatment/experimentation to prevent redistribution of Cu-MT

• Use freshly prepared fixatives

• Control fixation time carefully (10-15 minutes optimal for most applications)

• When studying copper-induced metallothionein expression, process control and experimental samples identically to allow accurate comparison

What controls should be included when using FITC-conjugated Cu-MT antibody?

A robust experimental design should include the following controls:

• Negative cellular control: Cells known not to express Cu-MT or MT-knockdown cells

• Isotype control: FITC-conjugated rabbit IgG at the same concentration as the primary antibody

• Unstained control: Cells processed without any antibody to establish autofluorescence baseline

• Positive control: Cells treated with copper sulfate (10-50 μM) to induce metallothionein expression

• Specificity control: Pre-absorption of antibody with purified Cu-MT protein to demonstrate binding specificity

• Blocking control: Cells incubated with blocking peptide prior to antibody application

These controls help distinguish true signal from background and non-specific binding, crucial for accurate data interpretation in fluorescence-based detection methods .

How can FITC-conjugated Cu-MT antibody be used to investigate subcellular localization of metallothionein under copper stress?

To investigate subcellular localization changes:

• Dual immunofluorescence approach:

  • Use FITC-conjugated Cu-MT antibody alongside organelle-specific markers (labeled with spectrally distinct fluorophores)

  • For mitochondrial localization, co-stain with MitoTracker Red

  • For lysosomal localization, co-stain with LysoTracker Red

• Time-course analysis:

  • Expose cells to varying copper concentrations (0.4 μM, 10 μM, 50 μM)

  • Fix cells at different timepoints (1, 3, 6, 12, 24 hours)

  • Analyze changes in Cu-MT distribution pattern

• Confocal microscopy analysis:

  • Use z-stack imaging to create 3D reconstructions of Cu-MT distribution

  • Perform colocalization analysis with organelle markers using appropriate software

  • Calculate Pearson's correlation coefficient to quantify degree of colocalization

Research indicates that metallothioneins affect the subcellular localization of accumulated copper in the cytoplasm, and this is particularly important when extracellular copper reaches physiological levels (10 μM) .

How can Cu-MT antibody be used to investigate the relationship between metallothionein expression and oxidative stress?

To investigate this relationship:

• Combined oxidative stress markers and Cu-MT detection:

  • Treat cells with copper and oxidative stress inducers (H₂O₂, menadione)

  • Perform dual labeling with FITC-conjugated Cu-MT antibody and markers for oxidative damage (8-OHdG antibody or CellROX dyes)

  • Analyze correlation between Cu-MT expression and oxidative damage indicators

• Functional studies in MT-knockout models:

  • Compare wild-type and MT-knockout cells' response to copper exposure

  • Quantify Cu-MT expression using FITC-conjugated antibody via flow cytometry

  • Measure oxidative stress parameters (ROS levels, lipid peroxidation, GSH/GSSG ratio)

• Gene expression correlation analysis:

  • Monitor Cu-MT protein levels using FITC-conjugated antibody

  • Simultaneously analyze expression of oxidative stress-responsive genes (SOD1, Ccs1)

  • Establish temporal relationships between Cu-MT induction and oxidative stress response

Research demonstrates that MT null cells fail to show increased levels of mRNAs encoding MT I, SOD1 (superoxide dismutase 1) and Ccs1 (copper chaperone for SOD) in response to copper exposure, suggesting metallothionein is necessary for the signaling pathway that induces gene expression in response to copper .

What are common issues when using FITC-conjugated Cu-MT antibody and how can they be resolved?

When optimizing protocols, it's recommended to perform systematic titration experiments to determine the optimal antibody concentration for your specific application and cell type .

How can the specificity of Cu-MT detection be verified in complex biological samples?

To verify antibody specificity:

• Competitive inhibition assay:

  • Pre-incubate FITC-conjugated Cu-MT antibody with purified Cu-MT protein at increasing concentrations

  • Apply to samples and assess signal reduction

  • A concentration-dependent decrease in signal indicates specific binding

• Western blot correlation:

  • Compare FITC immunofluorescence intensity with band intensity from Western blot

  • Samples with higher fluorescence should show stronger bands at the expected molecular weight (~5-8 kDa for MT)

• Genetic validation:

  • Use siRNA knockdown or CRISPR-Cas9 to reduce MT expression

  • Confirm reduced expression via RT-PCR

  • Verify corresponding decrease in FITC signal

• Copper induction experiments:

  • Treat cells with increasing copper concentrations to induce MT expression

  • Verify dose-dependent increase in FITC signal

  • Parallel analysis of MT mRNA levels should show correlation with protein detection

Studies have shown that metallothionein expression is strongly induced by copper exposure, and this induction can be detected at both mRNA and protein levels .

How does FITC-conjugated Cu-MT antibody compare with other metallothionein detection methods?

For studies requiring both spatial and quantitative information, combining FITC-conjugated antibody detection with image analysis software can provide semi-quantitative data on both Cu-MT expression levels and subcellular distribution .

What are the technical considerations when using FITC-conjugated Cu-MT antibody for multiplexed fluorescence microscopy?

For multiplexed imaging:

• Spectral compatibility considerations:

  • FITC emission (peak ~519 nm) must be sufficiently separated from other fluorophores

  • Compatible partners include TRITC/Cy3 (red), Cy5 (far-red), and DAPI (blue)

  • Avoid PE, which has significant spectral overlap with FITC

• Sequential acquisition strategy:

  • Acquire FITC channel first (before significant photobleaching occurs)

  • Use narrow bandpass filters to minimize bleed-through

  • Consider linear unmixing algorithms for closely overlapping spectra

• Cross-reactivity prevention:

  • When using multiple antibodies, select those raised in different host species

  • Include appropriate blocking steps between applications

  • Validate antibody specificity individually before multiplexing

• Antibody concentration balancing:

  • Titrate each antibody separately to determine optimal concentration

  • Adjust concentration ratios to achieve balanced signal intensity

  • Consider differences in antigen abundance when optimizing concentrations

When studying metallothionein's role in copper homeostasis, multiplexed approaches can reveal relationships between Cu-MT expression and distribution of copper transporters or chaperones .

How is FITC-conjugated Cu-MT antibody being used to advance understanding of metallothionein in pathogenic organisms?

Recent studies have employed fluorescent Cu-MT antibodies to investigate:

• Mycobacterium tuberculosis copper defense mechanisms:

  • FITC-conjugated antibodies targeting MymT (mycobacterial metallothionein) have revealed that this copper-binding protein can sequester up to 6 Cu(I) ions

  • Immunofluorescence studies showed that copper, cadmium, and compounds generating nitric oxide or superoxide induced MymT expression up to 1000-fold

  • The protein was found to bind copper within M. tuberculosis and partially protect the bacteria from copper toxicity

  • Comparative studies between wild-type and ΔmymT mutants demonstrated increased copper sensitivity in mutants lacking this metallothionein

• Yeast metallothionein mechanisms:

  • Fluorescence microscopy using tagged CUP1 (yeast metallothionein) has revealed that this protein enters mitochondria

  • Studies suggest metallothionein may limit concentrations of low-molecular-mass copper complexes in organelles

  • Spectroscopic analysis showed that fully-loaded CUP1 contained eight copper ions bound in a solvent-shielded core

These findings suggest metallothioneins play critical roles in microbial pathogenesis and stress response, opening new avenues for therapeutic intervention.

What emerging applications combine Cu-MT antibody detection with other advanced imaging techniques?

Emerging research applications include:

• Super-resolution microscopy:

  • STORM/PALM techniques combined with FITC-conjugated Cu-MT antibodies achieve ~20 nm resolution

  • This allows visualization of Cu-MT clusters and potential interaction with specific cellular structures

  • Nanoscale distribution patterns reveal previously unknown organizational principles of Cu-MT in response to stress

• Correlative light and electron microscopy (CLEM):

  • FITC signal from Cu-MT antibody guides subsequent electron microscopy analysis

  • Allows precise ultrastructural localization of Cu-MT in relation to cellular components

  • Particularly valuable for studying Cu-MT association with membranes and organelles

• Intravital microscopy:

  • FITC-conjugated Cu-MT antibodies adapted for in vivo imaging in transparent model organisms

  • Permits real-time monitoring of metallothionein expression during developmental processes

  • Enables visualization of Cu-MT dynamics during experimental manipulation of copper levels

• Live-cell FRET sensors:

  • Combining FITC-Cu-MT antibody fragments with complementary fluorophore-tagged copper chaperones

  • Allows monitoring of dynamic interactions between metallothionein and copper transport machinery

  • Provides real-time data on copper transfer mechanisms in living cells

These advanced techniques are helping to establish comprehensive models of how metallothioneins function in cellular copper homeostasis mechanisms .