CTR1 Antibody

What is CTR1 and why is it important in biological research?

CTR1 (Copper Transporter 1, also known as SLC31A1) is a high-affinity copper (Cu+) importer that is structurally and functionally conserved in yeast, plants, fruit flies, and humans . This protein provides the primary avenue for copper uptake in mammalian cells, thereby affecting copper homeostasis and embryonic development . CTR1 plays a crucial role in normal growth, development, cognition, and neurological function, as copper serves as a cofactor for many essential enzymes . Additionally, CTR1 has been implicated in the transport of platinum-based chemotherapeutic agents like cisplatin, making it relevant to cancer research and drug resistance studies .

What are the challenges in developing reliable CTR1 antibodies?

Developing reliable antibodies against CTR1 has proven particularly difficult, with polyclonal antibodies from different laboratories yielding conflicting results . This inconsistency has complicated the accurate identification and quantification of CTR1 in research settings. For example, contradictory findings have been reported regarding CTR1 localization in intestinal epithelial cells, with some studies suggesting basolateral membrane localization while others demonstrate apical localization . These discrepancies highlight the critical need for well-characterized, specific antibodies with validated epitope recognition.

What is the molecular structure of CTR1 and which epitopes are commonly targeted by antibodies?

CTR1 has a complex topology consisting of an approximately 56-amino acid amino-terminal extracellular domain (for plasma membrane-localized CTR1), an approximately 53-residue cytoplasmic loop, three transmembrane domains, and an approximately 15-amino acid cytosolic tail . Different antibodies target various epitopes within this structure. For example:

• The rabbit monoclonal antibody from Epitomics/Abcam (catalog #5773-1/AB129067) targets amino acids 1-30 at the N-terminal end of hCTR1

• Some polyclonal antibodies have been raised against peptides derived from the cytosolic loop of human CTR1

The choice of epitope can significantly impact antibody specificity and the detection of different forms of the protein.

What is the optimal sample preparation method for CTR1 detection by Western blotting?

For optimal Western blot detection of CTR1, post-nuclear membrane (PNM) preparations are strongly recommended over whole cell lysates (WCL) . When using whole cell lysates, the anti-CTR1 antibody detects various bands, making accurate quantification difficult . The recommended protocol involves:

• Isolating post-nuclear membranes from cells or tissues

• Solubilizing the membranes with appropriate detergents (e.g., n-dodecyl-β-D-maltoside)

• Using a 1:1000 dilution of the primary antibody for Western blotting

• Blocking with 5% powdered milk containing 0.1% Tween-20

This approach typically reveals three distinct bands: a 28 kDa band (a cross-reacting protein), a 33-35 kDa band (glycosylated hCTR1 monomer), and a 62-64 kDa band (possibly a dimeric form of CTR1) .

How can researchers confirm the specificity of CTR1 antibody signals?

To validate the specificity of CTR1 antibody signals, researchers should employ multiple complementary approaches:

• Compare signals between wild-type cells and CTR1 knockdown/knockout cells (e.g., using lentiviral-based short-hairpin RNA transduction with vectors like CTR1 shRNA Clone ID# TRCN0000043348)

• Utilize cells expressing tagged versions of CTR1 (e.g., myc-tagged hCTR1) and perform parallel detection with both anti-CTR1 and anti-tag antibodies

• Conduct immunoprecipitation experiments followed by Western blot analysis

• Perform surface biotinylation assays to confirm membrane localization

• Carry out deglycosylation experiments to verify glycosylated forms

When analyzing PNM preparations, evidence suggests that the 33-35 kDa and 62-64 kDa bands represent true CTR1 signals, while the 28 kDa band is a cross-reacting protein of unknown identity .

What controls should be included when using CTR1 antibodies for immunohistochemistry?

For immunohistochemical detection of CTR1, appropriate controls are essential to ensure signal specificity and accurate localization. Key controls include:

• Tissue from genetic knockout models, such as intestinal epithelial cell-specific Ctr1 knockout mice (Ctr1ᵢₙₜ/ᵢₙₜ)

• Parallel staining with antibodies against proteins with established localization patterns (e.g., hephaestin for basolateral membrane)

• Secondary antibody-only controls to assess non-specific binding

• Peptide competition assays to confirm epitope specificity

For example, in studies of intestinal CTR1 localization, samples from Ctr1ᵢₙₜ/ᵢₙₜ mice showed very little anti-CTR1 antibody immunoreactivity, confirming the specificity of the signal observed in control mice .

What are the different molecular weight forms of CTR1 detected by antibodies?

CTR1 antibodies detect several distinct molecular weight species that represent different forms of the protein:

Additionally, a 28 kDa band is frequently observed but represents a cross-reacting protein rather than CTR1 . The varying molecular weights reported in different studies may reflect differences in experimental conditions, cell types, or species variations.

How do post-translational modifications affect CTR1 detection?

Post-translational modifications, particularly glycosylation, significantly impact CTR1 detection and should be considered when interpreting antibody signals:

• CTR1 undergoes both O- and N-linked glycosylation, resulting in the mature 33-37 kDa species

• The glycosylation state of CTR1 can change in response to copper availability, representing a time-dependent, copper-specific posttranslational response

• Different glycoforms may exhibit altered antibody reactivity, trafficking patterns, or stability

• Deglycosylation experiments can help confirm the identity of glycosylated bands and distinguish them from cross-reacting proteins

Understanding these modifications is particularly important when studying CTR1 regulation in response to changing copper levels or other experimental conditions.

How can researchers differentiate between monomeric and dimeric forms of CTR1?

To distinguish between monomeric and potential dimeric forms of CTR1:

• Use non-reducing versus reducing conditions during sample preparation to preserve or disrupt disulfide bonds

• Perform crosslinking experiments to stabilize protein-protein interactions

• Employ gradient gels for better separation of proteins of different molecular weights

• Apply different detergents to solubilize membrane proteins while preserving or disrupting protein complexes

In Western blot analysis of PNM preparations, the 33-35 kDa band is consistent with the glycosylated CTR1 monomer, while the 62-64 kDa band likely represents a dimeric form . Multiple lines of evidence, including immunoprecipitation and surface biotinylation studies, support the assignment of these specific molecular weight bands as genuine CTR1 species .

How is CTR1 localized in intestinal epithelial cells?

The localization of CTR1 in intestinal epithelial cells has been a subject of debate. Recent comprehensive studies using immunohistochemistry have definitively demonstrated that CTR1 localizes to the apical membrane in intestinal epithelial cells of mouse, rat, and pig . This finding contradicts earlier reports suggesting exclusive basolateral localization in these cells .

Key evidence supporting apical localization includes:

• Immunohistochemical analysis of jejunal sections from control mice (Ctr1ᶠˡᵒˣ/ᶠˡᵒˣ) showing predominant CTR1 immunoreactivity at the apical cell surface

• Minimal immunoreactivity in intestinal epithelial cell-specific Ctr1 knockout mice (Ctr1ᵢₙₜ/ᵢₙₜ), confirming antibody specificity

• Parallel staining for hephaestin confirming its expected basolateral localization

• Consistent findings across three mammalian species

This apical localization is consistent with CTR1's role in dietary copper absorption from the intestinal lumen.

How does copper availability affect CTR1 expression and localization?

Copper availability has significant effects on CTR1 expression and localization, with important implications for copper homeostasis:

• Biotinylation of intestinal luminal proteins from mice fed a control or copper-deficient diet revealed elevated levels of both total and apical membrane CTR1 protein in response to dietary copper limitation

• Experiments in cultured HEK293T cells demonstrated that alterations in the glycosylated form of CTR1 in response to copper availability were time-dependent and copper-specific posttranslational responses

• These changes in CTR1 localization at the apical membrane likely represent an adaptive response to homeostatically modulate copper absorption based on dietary availability

These findings suggest that cells can regulate copper uptake by controlling the abundance of CTR1 at the cell surface, particularly at the site of intestinal copper absorption.

What techniques are most effective for studying CTR1 trafficking?

Several complementary techniques are particularly effective for investigating CTR1 trafficking and localization:

• Immunohistochemistry and confocal immunofluorescence microscopy for visualizing CTR1 distribution in tissue sections or cultured cells

• Surface biotinylation for quantifying changes in cell surface CTR1 levels

• Cell fractionation to separate different cellular compartments and analyze CTR1 distribution

• Live-cell imaging using tagged CTR1 constructs to monitor trafficking in real-time

• Electron microscopy for ultrastructural localization studies

Each approach has strengths and limitations, and combining multiple techniques provides the most robust evidence for CTR1 localization and trafficking dynamics. For instance, both immunohistochemistry and biotinylation approaches have been used together to establish the apical localization of CTR1 in intestinal epithelial cells and its regulation in response to copper availability .

How is CTR1 involved in cisplatin transport and resistance?

CTR1 plays a significant role in the transport of cisplatin, a widely used cancer chemotherapeutic agent:

• CTR1 is a high-affinity copper influx transporter that also mediates the influx of cisplatin

• Loss of CTR1 expression has been implicated in the development of resistance to cisplatin

• Studies using CTR1 knockdown approaches have demonstrated altered cisplatin accumulation and sensitivity

• Understanding CTR1 regulation may provide insights into mechanisms of platinum drug resistance and potential strategies to overcome it

This dual role of CTR1 in both copper and cisplatin transport connects copper homeostasis with cancer treatment efficacy and resistance mechanisms .

What approaches can be used to study CTR1-mediated drug transport?

To investigate CTR1-mediated drug transport, researchers can employ several experimental approaches:

• Generate stable cell lines with modified CTR1 expression:

  • Overexpression systems using lentiviral vectors (e.g., pLenti6/V5-DEST)

  • Knockdown systems using shRNA (e.g., CTR1 shRNA Clone ID# TRCN0000043348)

  • CRISPR-Cas9 knockout or knock-in systems

• Measure drug accumulation and sensitivity:

  • Quantify intracellular platinum accumulation

  • Assess cell survival and cytotoxicity profiles

  • Correlate CTR1 expression levels with drug response

• Analyze CTR1 expression and localization:

  • Monitor changes in response to drug exposure

  • Evaluate surface versus intracellular distribution

  • Examine post-translational modifications

These approaches allow researchers to dissect the mechanistic basis of CTR1-mediated drug transport and identify factors that influence drug efficacy and resistance.

How can CTR1 antibodies be used in copper-related disease research?

CTR1 antibodies are valuable tools for investigating copper-related diseases:

• In Menkes and Wilson's diseases (disorders of copper metabolism):

  • Assess CTR1 expression levels and localization in patient samples

  • Investigate potential compensatory changes in copper transport systems

  • Evaluate effects of therapeutic interventions on CTR1 expression

• In neurodegenerative conditions with copper dysregulation:

  • Examine CTR1 distribution in brain regions affected by Alzheimer's or Parkinson's disease

  • Correlate CTR1 levels with copper content and disease markers

  • Test effects of copper chelation or supplementation on CTR1 regulation

• In developmental disorders:

  • Monitor CTR1 expression during embryonic development

  • Assess the impact of copper deficiency on CTR1 distribution and function

These applications provide insights into disease mechanisms and potential therapeutic approaches targeting copper homeostasis.

What are common challenges in CTR1 Western blotting and how can they be addressed?

Researchers frequently encounter several challenges when performing Western blotting for CTR1:

The rabbit monoclonal antibody against the N-terminal of hCTR1 has been well-characterized and allows for reliable identification and quantification of hCTR1 when used with appropriate controls and preparation methods .

How can immunoprecipitation of CTR1 be optimized?

For successful immunoprecipitation of CTR1, the following optimized protocol has been validated:

• Sample preparation:

  • Prepare post-nuclear membranes from cells or tissues

  • Resuspend in 0.1 M phosphate (pH 7.2), 150 mM NaCl, 5 mM dithiothreitol, and 1% n-dodecyl-β-D-maltoside

  • Incubate for 1 hour at room temperature for solubilization

• Immunoprecipitation procedure:

  • Dilute solubilized samples in IP buffer (50 mM phosphate, pH 7.2, 200 mM NaCl, 2.5 mM dithiothreitol, and 0.5% n-dodecyl-β-D-maltoside)

  • Pre-clear with protein A/G plus agarose beads

  • Add anti-CTR1 antibody at 1:100 dilution and rotate at 4°C for 60 minutes

  • Add protein A/G plus agarose beads and rotate overnight at 4°C

• Analysis:

  • Elute proteins and analyze by Western blotting with anti-CTR1 antibody

  • Include controls such as anti-transferrin receptor for loading

This approach has been successfully used to confirm the identity of CTR1 bands detected by Western blotting.

What strategies can resolve conflicting results in CTR1 localization studies?

To address conflicts in CTR1 localization studies, researchers should implement the following strategies:

• Use multiple, well-characterized antibodies targeting different epitopes

• Include genetic controls like CTR1 knockout or knockdown samples

• Employ complementary localization techniques:

  • Immunohistochemistry on properly fixed tissues

  • Confocal immunofluorescence microscopy

  • Surface biotinylation

  • Subcellular fractionation

• Consider species, tissue, and cell-type specific differences:

  • Validate findings across multiple species (e.g., mouse, rat, pig)

  • Assess potential differences between cell culture models and in vivo tissues

  • Examine effects of experimental conditions on localization

This multi-faceted approach has resolved previous controversies regarding CTR1 localization in intestinal epithelial cells, definitively establishing its apical membrane expression across multiple mammalian species .

How are new antibody technologies enhancing CTR1 research?

Recent advances in antibody technology are significantly improving CTR1 research capabilities:

• Monoclonal antibody development:

  • The rabbit monoclonal antibody against the N-terminal of hCTR1 represents a significant advance, enabling reliable identification and quantification of hCTR1

  • Monoclonal antibodies provide greater consistency and specificity compared to polyclonal alternatives

• Enhanced detection methods:

  • Super-resolution microscopy for nanoscale localization studies

  • Multiplexed immunofluorescence for simultaneous detection of CTR1 with other proteins

  • Proximity labeling techniques to identify CTR1-interacting proteins

• Application-specific modifications:

  • Directly conjugated antibodies for flow cytometry and immunofluorescence

  • Fragment-based antibodies for improved tissue penetration

  • Recombinant antibody technology for consistent production

These technologies are addressing previous limitations in CTR1 research and opening new avenues for investigation.

What are potential applications of CTR1 antibodies in precision medicine?

CTR1 antibodies hold promise for several applications in precision medicine:

• Predictive biomarkers for platinum-based chemotherapy:

  • Assessing CTR1 expression in tumor biopsies to predict cisplatin response

  • Monitoring changes in CTR1 levels during treatment to detect emerging resistance

• Copper homeostasis assessment:

  • Evaluating CTR1 expression in disorders of copper metabolism

  • Guiding personalized copper supplementation or chelation strategies

• Targeted drug delivery:

  • Developing antibody-drug conjugates targeting CTR1-expressing cells

  • Creating nanoparticle formulations with enhanced CTR1-mediated uptake

• Companion diagnostics:

  • Pairing CTR1 expression analysis with emerging therapies targeting copper-dependent processes

  • Identifying patients most likely to benefit from specific therapeutic approaches

As our understanding of CTR1 biology continues to evolve, these applications may become increasingly relevant to clinical practice.

What future research directions are emerging in CTR1 antibody applications?

Several promising research directions are emerging for CTR1 antibody applications:

• Single-cell analysis:

  • Investigating cell-to-cell variability in CTR1 expression and localization

  • Correlating CTR1 levels with functional parameters at the single-cell level

• Structural studies:

  • Using conformation-specific antibodies to probe CTR1 structural states

  • Developing antibodies recognizing specific CTR1 complexes or oligomeric forms

• Dynamic regulation:

  • Real-time imaging of CTR1 trafficking using tagged antibody fragments

  • Monitoring acute responses to changing copper levels or drug exposures

• Therapeutic targeting:

  • Developing function-modulating antibodies to enhance or inhibit CTR1 activity

  • Exploiting CTR1 for targeted delivery of therapeutic agents

• Cross-species comparisons:

  • Extending the validated apical localization of intestinal CTR1 to additional species

  • Investigating evolutionary conservation and divergence in CTR1 regulation

These emerging directions promise to further expand our understanding of CTR1 biology and its therapeutic potential.