COPT1 Antibody

What is COPT1 and what cellular roles does it play?

COPT1, also known as high-affinity copper uptake protein 1 (hCTR1), Solute carrier family 31 member 1 (SLC31A1), or CTR1, is a plasma membrane protein involved in high-affinity copper uptake across cellular membranes. This protein plays a major role in copper translocation into the cytoplasm in eukaryotes . COPT1 is essential for maintaining proper copper homeostasis, which is critical for numerous biological processes including mitochondrial respiration, antioxidant defense, and iron metabolism. The protein functions as a homotrimer to effect the uptake of dietary copper . In plants, COPT1 participates in copper uptake through root tips, and studies in Arabidopsis show that altered COPT1 expression affects development, including hypocotyl growth and flowering time .

What is the structure and cellular localization of COPT1?

COPT1 is a multi-pass membrane protein containing three transmembrane domains . The human COPT1 protein is 190 amino acids in length. Its N-terminal domain contains over 60 amino acid residues and is predicted to contain an intrinsically disordered sequence with a high propensity for misfolding . The protein predominantly localizes to the plasma membrane in various cell types, similar to yeast CTR1 . In intestinal epithelial cells, COPT1 specifically localizes to the apical membrane . Post-translational modifications include O-glycosylation at Thr-27, which protects the protein from proteolytic cleavage in the N-terminal extracellular domain .

How is COPT1 expression regulated in different tissues?

COPT1 is expressed in all examined organs and tissues, with particularly high levels in the liver and kidney . Additional expression has been documented in brain, lung, skeletal muscle, and skin tissues . The subcellular localization patterns may vary depending on the cell type, which might be associated with cell-type specific dynamics of COPT1 secretion or recycling between the plasma membrane and intracellular compartments . Studies in Arabidopsis show that COPT1 expression can be affected by light conditions and may interact with circadian rhythms .

What types of COPT1 antibodies are available for research?

Several types of COPT1 antibodies are available for research applications, including:

• Monoclonal antibodies specific to endogenous COPT1 protein, such as the mouse monoclonal IgG described in the search results

• Antibodies against epitope-tagged COPT1 constructs, including:

  • Anti-myc antibodies for detection of myc-tagged COPT1

  • Anti-HA antibodies for detection of HA-tagged COPT1

The choice between these options depends on the specific research question and experimental system. Monoclonal antibodies against endogenous COPT1 are advantageous for studying the native protein without potential artifacts from overexpression or tagging, while epitope-tagged constructs offer high specificity and consistent detection across different experimental conditions.

How can I validate the specificity of my COPT1 antibody?

Validating antibody specificity is crucial for reliable research results. For COPT1 antibodies, consider these validation approaches:

• Knockout/knockdown controls: Compare antibody signal between wild-type samples and those where COPT1 expression is reduced or eliminated. The search results describe COPT1 knockout mice and Arabidopsis studies that could serve as negative controls .

• Overexpression controls: Compare antibody signal in samples overexpressing COPT1 versus control samples. Several studies in the search results use overexpression systems that could serve as positive controls .

• Peptide competition assay: Pre-incubate the antibody with the immunogenic peptide (if available) before application to samples. The search results mention a COPT1 monoclonal antibody derived from a synthesized peptide region of human protein .

• Cross-reactivity tests: Test the antibody on samples from different species to confirm the expected reactivity pattern. The monoclonal antibody in search result shows reactivity to both human and mouse COPT1.

• Molecular weight verification: Confirm that the detected band appears at the expected molecular weight (~20 kDa for COPT1) .

What are the optimal conditions for Western blot detection of COPT1?

For optimal Western blot detection of COPT1, consider the following parameters based on the search results:

• Antibody dilution: For the monoclonal antibody described, a dilution range of 1:500-1:2000 is recommended for Western blot applications .

• Expected molecular weight: COPT1 appears as a band of approximately 20 kDa on Western blots .

• Sample preparation: Given that COPT1 is a membrane protein, effective membrane protein extraction protocols should be employed. Complete solubilization with appropriate detergents is essential.

• Loading controls: When comparing COPT1 levels between samples, appropriate loading controls should be used. For plant studies, Rubisco large subunit has been used .

• Blocking and washing: Standard Western blot protocols should be followed, with optimization of blocking conditions to minimize background while maintaining specific signal.

The quality of protein extraction is particularly important when working with transmembrane proteins like COPT1, as incomplete solubilization can lead to protein aggregation and inconsistent results.

How can I study COPT1 protein stability and degradation?

Based on the search results, several approaches have been used to study COPT1 protein stability and degradation:

• Genetic approaches: The search results describe studies comparing COPT1 protein levels between wild-type and mutant backgrounds. For example, researchers observed significantly reduced COPT1 protein accumulation in UBAC2 mutants despite similar transcript levels, indicating post-transcriptional regulation .

• Signal peptide modifications: Researchers have experimented with altering the N-terminal structure of COPT1 by inserting signal peptides (e.g., PR1 signal peptide) and studying how these modifications affect protein stability and accumulation .

• Cross-genetic background comparisons: To accurately assess protein stability factors, researchers established experimental systems where the same transgene was integrated at identical genomic locations in different genetic backgrounds, enabling direct comparison of protein levels while controlling for positional effects of transgene integration .

• Transcript versus protein level correlation analysis: By simultaneously measuring both transcript and protein levels of COPT1 constructs, researchers can determine whether changes in protein abundance are due to transcriptional or post-transcriptional mechanisms .

What approaches can be used to study COPT1 subcellular localization?

Several approaches can be used to study COPT1 subcellular localization:

• Fluorescent protein fusions: The search results describe the use of GFP-tagged COPT1 constructs to visualize the protein's subcellular localization in living cells. This approach allows for real-time monitoring of protein distribution .

• Immunofluorescence microscopy: Using COPT1-specific antibodies for immunostaining fixed cells or tissues to visualize the native protein's distribution.

• Subcellular fractionation: Biochemical separation of cellular compartments followed by Western blot analysis with COPT1 antibodies to determine which fractions contain the protein.

• Co-localization studies: Combining COPT1 detection with markers for specific subcellular compartments to precisely define its localization.

The search results indicate that COPT1 primarily localizes to the plasma membrane, similar to yeast CTR1, but its distribution patterns might vary depending on cell type due to cell-specific dynamics of secretion or recycling between the plasma membrane and intracellular compartments .

How can I investigate COPT1 interactions with other proteins?

Based on the search results, several approaches have been successfully used to study COPT1 protein interactions:

• Yeast two-hybrid assays: This approach was used to identify and characterize the interaction between COPT1 and UBAC2a in Arabidopsis. Researchers created truncated versions of COPT1 to map specific interaction domains .

• Co-immunoprecipitation: Although not explicitly described in the search results, co-immunoprecipitation using COPT1 antibodies would be a standard approach to identify interacting protein partners in native conditions.

• Domain mapping through truncation analysis: The search results describe systematic truncation of COPT1 protein to identify critical regions for protein interactions. This revealed that a large N-terminal COPT1 sequence of more than 100 residues was required for interaction with UBAC2a .

• Genetic interaction studies: Comparing the phenotypes of single and double mutants can provide evidence for functional interactions. The search results describe studies with UBAC2 mutants and their effects on COPT1 protein accumulation .

• Structural analysis: Prediction of intrinsically disordered regions (as mentioned for the N-terminal domain of COPT1) can help identify potential protein interaction surfaces .

What experimental approaches can address potential artifacts in COPT1 antibody studies?

Potential artifacts in antibody-based studies of COPT1 can be addressed through several experimental approaches:

• Multiple antibody validation: Use different antibodies raised against distinct epitopes of COPT1 to confirm findings. This reduces the risk of epitope-specific artifacts.

• Complementary detection methods: Compare results from antibody-based detection (Western blot, immunofluorescence) with alternative approaches such as mass spectrometry-based proteomics.

• Tagged versus endogenous protein comparison: The search results describe various tagged versions of COPT1 (myc-tagged, GFP-tagged). Comparing results between studies of endogenous COPT1 and these tagged versions can reveal potential artifacts introduced by tags.

• Controlled expression systems: The search results describe both native promoter-driven expression and overexpression systems. Comparing results between these approaches can help identify artifacts related to protein expression levels .

• Genetic background controls: The search results emphasize the importance of controlling for genetic background effects by comparing plants with the same transgene integrated at the same genomic location but differing only in the zygosity of mutations in genes of interest .

How can COPT1 antibodies be used to study copper transport mechanisms?

COPT1 antibodies can be instrumental in studying copper transport mechanisms through several experimental approaches:

• Quantitative protein analysis: Western blot analysis using COPT1 antibodies allows researchers to quantify COPT1 protein levels under various conditions, such as copper deficiency or excess. This helps understand how COPT1 expression is regulated in response to copper availability.

• Protein modification studies: COPT1 antibodies can detect post-translational modifications that might regulate the protein's activity. The search results mention O-glycosylation at Thr-27 that protects COPT1 from proteolytic cleavage .

• Structure-function analyses: By combining COPT1 antibody detection with mutagenesis of specific protein domains, researchers can identify regions critical for copper transport function.

• Trafficking studies: COPT1 antibodies can be used to track protein localization and redistribution in response to changing copper levels or cellular signals.

• Comparative studies across tissues: The search results indicate that COPT1 is expressed in multiple tissues . Antibody-based detection can reveal tissue-specific expression patterns and potential specialized copper transport mechanisms.

• Protein complex analysis: Co-immunoprecipitation with COPT1 antibodies can identify proteins that associate with COPT1 during copper transport.

Why might I observe different COPT1 expression levels across seemingly identical samples?

The search results highlight several factors that can contribute to variability in COPT1 expression levels:

• Positional effects of transgene integration: The search results explicitly mention "substantial variation in the intensities of GFP signals even among independent lines of the same genetic background, which complicated the comparison." This variability was attributed to positional effects of transgene integration at different genome sites .

• Genetic background differences: The studies described in the search results went to considerable lengths to control for genetic background effects by generating plants with transgenes integrated at the same genomic site but differing only in specific mutations of interest .

• Transcriptional versus post-transcriptional regulation: The search results describe cases where COPT1 protein levels differed significantly despite similar transcript levels, indicating important post-transcriptional regulatory mechanisms .

• Promoter strength variations: Different studies used either native COPT1 promoters or strong constitutive promoters like CaMV 35S, each with distinct expression characteristics and variability patterns .

• Protein stability factors: The search results indicate that protein quality control mechanisms, particularly those involving UBAC2 proteins, significantly affect COPT1 accumulation .

What are common technical challenges when working with COPT1 antibodies?

Based on the search results and general knowledge about membrane protein antibodies, common technical challenges include:

• Membrane protein extraction efficiency: As a multi-pass membrane protein, COPT1 can be difficult to extract completely from cellular membranes, potentially leading to inconsistent detection.

• Protein aggregation: Membrane proteins like COPT1 can form aggregates during sample preparation, particularly when using inappropriate detergents or buffer conditions.

• Background signal in Western blots: The search results don't explicitly mention this issue, but membrane proteins often present challenges in achieving clean Western blot signals due to hydrophobic interactions.

• Post-translational modifications: The search results mention O-glycosylation of COPT1 , which could potentially affect antibody recognition and result in variable detection efficiency depending on the modification state.

• Protein degradation: The search results indicate that COPT1 stability is regulated by protein quality control mechanisms , which could lead to variable detection if samples are not prepared with appropriate protease inhibitors.

• Cross-reactivity with related proteins: The SLC31A family includes multiple members, and antibodies might cross-react with related proteins if not properly validated.