CUP1-1 Antibody

What is CUP1-1 and what is its biological function?

CUP1-1 (Copper metallothionein 1-1) is one of two identical genetic copies of the CUP1 gene in Saccharomyces cerevisiae (the other being CUP1-2). The CUP1 protein functions primarily to protect yeast cells against copper toxicity by tightly chelating copper ions. It may also act as a depository for copper designated for effective transfer into the apo forms of copper proteins .

Recent research has revealed that CUP1 is present in both cytosolic and mitochondrial compartments, with its abundance dependent on copper supplementation levels in growth media. Specifically, CUP1 has been detected in the intermembrane space of mitochondria, suggesting additional functions beyond cytosolic copper sequestration .

Beyond copper homeostasis, CUP1 has been shown to contribute to nitrosative stress tolerance in yeast, potentially by scavenging nitric oxide (NO). Experiments demonstrated that strains lacking CUP1 exhibited growth defects under nitrosative stress conditions, while CUP1 overexpression improved growth under these conditions .

What are the key specifications of CUP1-1 antibodies?

Commercial CUP1-1 antibodies typically have the following specifications:

The antibody is designed to recognize specific epitopes of the CUP1-1 protein, allowing researchers to detect, quantify, and localize this metallothionein in various experimental contexts .

How should I validate a CUP1-1 antibody before using it in my experiments?

Proper validation is critical before using any antibody in research applications. For CUP1-1 antibodies, follow these validation steps:

• Specificity testing: Compare staining/detection patterns between wild-type strains and cup1Δ strains (lacking both CUP1-1 and CUP1-2). A specific antibody should show signals in wild-type but not in the deletion strain .

• Western blot verification: Confirm the antibody detects a protein of the expected molecular weight (~7 kDa for native CUP1 or ~8.5 kDa for His-tagged versions). Note that CUP1 often appears as multiple bands due to its copper-binding properties and potential truncation of the first eight amino acid residues .

• Expression correlation: Verify that signal intensity increases when CUP1 is overexpressed or when cells are treated with copper, which induces CUP1 expression .

• Subcellular localization: If using the antibody for immunofluorescence, confirm that the staining pattern matches the expected subcellular distribution (both cytosolic and mitochondrial) .

• Cross-reactivity assessment: Test the antibody against other metallothioneins to ensure specificity for CUP1 versus other metal-binding proteins .

Remember that an antibody working in one application (e.g., Western blot) does not guarantee performance in another (e.g., immunofluorescence) .

How can I optimize CUP1-1 antibody use for studying copper-dependent protein expression?

Optimizing CUP1-1 antibody use for copper-dependent protein expression studies requires careful experimental design:

• Copper concentration gradient: Establish a dose-response curve by exposing yeast to varying copper concentrations (typically 0-1 mM CuSO₄) and measure CUP1-1 protein levels at each concentration. This helps determine the optimal copper concentration for maximum protein induction without cellular toxicity .

• Time-course analysis: Collect samples at multiple time points (0, 10, 20, 30, 60 minutes) following copper exposure to capture the dynamics of CUP1-1 expression and subsequent downregulation. RT-qPCR can be performed in parallel to correlate protein and mRNA levels .

• Subcellular fractionation: To differentiate between cytosolic and mitochondrial CUP1-1, implement subcellular fractionation protocols prior to immunoblotting. Enriched mitochondrial and cytosolic fractions should be analyzed separately to determine compartment-specific expression patterns .

• Dual detection strategy: When studying both CUP1-1 and CUP1-2 simultaneously, consider using GFP-tagged strains where one copy is tagged and the other remains native. This approach allows differentiation between the two identical copies through size differences in immunoblotting .

• Quantification controls: Include purified recombinant CUP1-1 protein standards on your Western blots to generate a standard curve for accurate quantification of native protein levels across different conditions .

For mitochondrial localization studies, mitoplasting techniques followed by protease protection assays can help determine precise submitochondrial localization, as CUP1 has been specifically localized to the intermembrane space of mitochondria .

What approaches can resolve discrepancies between CUP1-1 antibody detection and gene expression data?

When facing discrepancies between protein detection using CUP1-1 antibodies and gene expression data, consider these methodological approaches:

• Post-translational modification analysis: CUP1-1 protein may undergo modifications affecting antibody recognition without changing mRNA levels. Mass spectrometry can detect truncations, such as the documented removal of the first eight amino acids in mature CUP1 protein .

• Protein stability assessment: Perform cycloheximide chase experiments to determine if differences arise from altered protein stability rather than transcription/translation rates. CUP1 may have different half-lives depending on copper binding status and cellular compartment .

• Copper occupancy verification: Use chelator treatments to generate apo-CUP1 and compare antibody recognition of metalated versus non-metalated forms. Some antibodies may preferentially detect one form over the other, explaining detection inconsistencies .

• Cross-validation with multiple techniques:

  • Western blot with different antibodies targeting separate epitopes

  • Mass spectrometry for direct protein quantification

  • Fluorescence microscopy with tagged CUP1 variants

  • Chromatin immunoprecipitation (ChIP) to assess promoter activity

• Strain-specific variations: Test different yeast strain backgrounds (e.g., BY4741, W303, DTY005) as CUP1 detection patterns can vary between strains. Document differences systematically to determine if discrepancies are strain-dependent .

When analyzing ChIP data for CUP1, note the unique histone modification patterns where H3 acetylation increases during expression (10-20 minutes after copper exposure) while H4 acetylation decreases. During shutdown (around 30 minutes), these patterns reverse with decreased H3 acetylation and increased H4 acetylation .

How can I use CUP1-1 antibodies to study the relationship between copper metabolism and nitrosative stress?

Recent research has established connections between CUP1 function and nitrosative stress tolerance. To investigate this relationship using CUP1-1 antibodies:

• Combined stress experiments: Expose yeast cultures to both copper (CuSO₄) and nitrosative stress agents (acidified nitrite or NO donors like NOC-5) simultaneously or sequentially. Monitor CUP1-1 protein levels using the antibody under these various stress conditions .

• In vitro NO scavenging assay: Use purified CUP1 protein (immunoprecipitated with your CUP1-1 antibody) and fluorescent NO probes like DAF-FM to directly measure NO scavenging capacity of the protein. Compare wild-type and mutant versions of CUP1 to identify key residues involved in NO interactions .

• Immunoprecipitation-based interaction studies: Employ CUP1-1 antibodies to pull down CUP1 complexes under different stress conditions to identify potential protein partners involved in both copper and nitrosative stress responses. Mass spectrometry analysis of co-precipitated proteins can reveal novel interactors .

• Subcellular redistribution analysis: Use immunofluorescence with CUP1-1 antibodies to track potential stress-induced relocalization of CUP1 between cytosol and mitochondria. This can be particularly important as mitochondrial CUP1 may have distinct roles in managing nitrosative stress compared to cytosolic CUP1 .

• Genetic interaction mapping: In strains with varying CUP1 expression levels (deletion, wild-type, overexpression), use the antibody to correlate CUP1 protein levels with survival under nitrosative stress. This approach can identify threshold effects and establish quantitative relationships between protein abundance and stress tolerance .

For flow cytometry experiments measuring intracellular NO levels in conjunction with CUP1 detection, proper compensation controls are essential when using multiple fluorophores to prevent false correlations due to spectral overlap .

What are the optimal conditions for using CUP1-1 antibodies in different applications?

Each application requires specific optimization of CUP1-1 antibody conditions:

Western Blotting:

• Sample preparation: Include metal chelators (e.g., EDTA) in lysis buffers to maintain protein integrity

• Recommended dilution: Typically 1:1000-1:2000 for most commercial antibodies

• Blocking solution: 5% non-fat milk in TBST is generally effective

• Special considerations: Use reducing conditions with β-mercaptoethanol to ensure proper protein denaturation

• Controls: Include cup1Δ strain lysates and CUP1 overexpression samples

Immunoprecipitation:

• Binding conditions: Use gentle lysis buffers (e.g., 1% NP-40, 150mM NaCl, 50mM Tris pH 7.5) to preserve protein interactions

• Antibody amount: Typically 2-5 μg per sample

• Preclearance: Recommended to reduce background

• Beads: Protein G-coupled beads for most rabbit IgG antibodies

• Elution: Consider non-denaturing elution for functional studies of copper-bound CUP1

Immunofluorescence:

• Fixation: 4% paraformaldehyde is recommended

• Permeabilization: 0.1% Triton X-100

• Antibody dilution: Start with 1:100, then optimize

• Counterstains: Include mitochondrial markers (e.g., MitoTracker) for colocalization studies

• Controls: Use CUP1-GFP strains for verification of staining patterns

Chromatin Immunoprecipitation (ChIP):

• Crosslinking: 1% formaldehyde for 10 minutes at room temperature

• Sonication: Optimize to achieve 200-500 bp DNA fragments

• Antibody amount: 5-10 μg per sample

• Washing stringency: Increase salt concentration in wash buffers to reduce background

• Analysis: Use qPCR with primers specific to the CUP1 promoter, coding region, and regions upstream of RUF5

For all applications, perform initial antibody titration experiments to determine the optimal concentration that maximizes specific signal while minimizing background.

What controls should I include when using CUP1-1 antibodies?

Proper controls are essential for reliable interpretation of results with CUP1-1 antibodies:

Genetic Controls:

• Negative control: cup1Δ strain (deletion of both CUP1-1 and CUP1-2) should show no signal

• Positive control: CUP1 overexpression strain should show enhanced signal

• Induction control: Wild-type cells treated with copper (1 mM CuSO₄) to induce CUP1 expression

• Specificity control: Cells expressing only one of the two CUP1 copies (either CUP1-1 or CUP1-2)

Technical Controls:

• Primary antibody omission: To assess secondary antibody non-specific binding

• Isotype control: Use non-specific IgG from the same host species at matching concentration

• Pre-absorption control: Pre-incubate antibody with purified CUP1 protein to confirm specificity

• Dilution series: Test multiple antibody concentrations to determine optimal signal-to-noise ratio

Application-Specific Controls:

• For Western blot: Include molecular weight markers and recombinant CUP1 protein standards

• For immunofluorescence: Include subcellular markers (e.g., mitochondrial, cytosolic markers)

• For flow cytometry: Include fluorescence minus one (FMO) controls for proper gating

• For ChIP: Include input samples, no-antibody controls, and positive control regions

Physiological Controls:

• Metal depletion: Cells grown in copper-depleted media should show reduced CUP1 levels

• Time course: Samples collected at different times after copper induction to capture expression dynamics

• Stress conditions: Compare copper stress to other metal stresses to confirm specificity of response

When analyzing results, compare signal intensities across all controls to ensure proper interpretation and avoid false positives or negatives.

How can I troubleshoot common issues with CUP1-1 antibodies?

When encountering problems with CUP1-1 antibody experiments, consider these troubleshooting approaches:

No or Weak Signal:

• Protein expression check: Verify CUP1 expression using copper induction or RT-qPCR

• Antibody concentration: Increase antibody concentration or incubation time

• Epitope accessibility: Try different sample preparation methods (native vs. denaturing)

• Detection system: Use more sensitive detection methods (ECL Prime vs. standard ECL)

• Protein extraction: Ensure your lysis buffer effectively extracts CUP1 (which may be bound to membranes)

High Background:

• Blocking optimization: Test different blocking agents (milk vs. BSA) and increase blocking time

• Antibody dilution: Use higher dilutions of primary and secondary antibodies

• Wash stringency: Increase wash duration and buffer stringency (add more salt or detergent)

• Cross-reactivity: Pre-absorb antibody with yeast lysate from cup1Δ strain

• Secondary antibody: Test alternative secondary antibodies with lower cross-reactivity

Multiple Bands or Unexpected Size:

• Protein degradation: Add protease inhibitors to all buffers

• Post-translational modifications: CUP1 is known to undergo N-terminal truncation (first 8 amino acids)

• Copper binding status: Different copper-binding states may affect migration pattern

• Cross-reactivity: Confirm specificity using cup1Δ strain and recombinant protein

• Aggregation: Add reducing agents to disrupt potential disulfide bonds

Subcellular Localization Discrepancies:

• Fractionation purity: Verify compartment separation with markers (e.g., porin for mitochondria)

• Fixation artifacts: Test different fixation methods that preserve CUP1 localization

• Copper levels: Vary copper concentrations as localization may be copper-dependent

• Strain differences: Compare results across different yeast strain backgrounds

Quantification Challenges:

• Saturation: Ensure signal is within linear range of detection

• Normalization: Use multiple loading controls (e.g., actin, total protein)

• Standardization: Include purified CUP1 protein at known concentrations

• Reproducibility: Perform biological triplicates and calculate variation

For technical issues specific to flow cytometry analyses with CUP1-1 antibodies, follow guidelines for selecting appropriate fluorophores based on antigen density, use compensation beads, and include FMO controls for proper gating .

How can I develop a ChIP protocol using CUP1-1 antibodies to study histone modifications at the CUP1 locus?

Developing a ChIP protocol for studying histone modifications at the CUP1 locus requires careful optimization:

• Experimental Design:

  • Include time points before copper addition (0 min) and at 10, 20, 30, and 60 minutes after exposure

  • Prepare parallel samples for RNA extraction to correlate ChIP results with transcription levels

  • Use 1 mM CuSO₄ as a standard induction concentration

• ChIP Protocol Steps:

  • Fixation: Cross-link proteins to DNA with 1% formaldehyde for 10 minutes at room temperature

  • Sonication: Optimize sonication conditions to achieve DNA fragments of 200-500 bp

  • Immunoprecipitation: Use antibodies against specific histone modifications (H3ac, H4ac) and unmodified histones as controls

  • DNA isolation: Purify DNA after reversing cross-links

  • Analysis: Perform qPCR with primers targeting upstream promoter, CUP1 coding region, and region upstream of RUF5

• Primer Design for qPCR:

  • CUP1 promoter region: -250 to -150 relative to start codon

  • CUP1 coding region: +50 to +150

  • RUF5 upstream region: Design based on the antisense strand

  • Control region: Use a region not expected to show histone modifications during copper exposure

• Key Histone Modifications to Target:

  • H3 acetylation (increases during activation, decreases during shutdown)

  • H4 acetylation (decreases during activation, increases during shutdown)

  • Additional modifications: H3K4me3, H3K36me3 (associated with active transcription)

• Data Interpretation:

  • Normalize ChIP data to input DNA and unmodified histone levels

  • Compare modification patterns across all time points

  • Correlate with mRNA levels to establish temporal relationships

  • Analyze both CUP1 and RUF5 regions simultaneously to detect potential coordinated regulation

• Advanced Analysis:

  • Identify specific lysine acetyltransferases (KATs) and histone deacetylases (HDACs) involved by using strains with deletions of candidate enzymes

  • Perform sequential ChIP (re-ChIP) to detect co-occurring modifications

  • Combine with CUP1-1 antibody ChIP to correlate protein binding with histone modifications

This protocol leverages the unique histone modification patterns at the CUP1 locus, where, unlike many genes, H3 and H4 acetylation show inverse patterns during activation and shutdown .

What new roles for CUP1 have been discovered beyond copper homeostasis?

Recent research has uncovered several unexpected functions of CUP1 beyond its classical role in copper detoxification:

• Nitrosative Stress Response: CUP1 has been identified as a contributor to nitrosative stress tolerance in yeast. Deletion of CUP1 genes resulted in growth defects under nitrosative stress conditions, while overexpression improved growth. Mechanistically, CUP1 appears to function as an NO scavenger, directly reducing intracellular NO levels .

• Mitochondrial Localization: CUP1 has been detected in the intermembrane space of mitochondria across multiple yeast strains (W303, DTY005, BY4741), suggesting a previously unrecognized role in mitochondrial function. This localization was confirmed through multiple approaches including liquid chromatography, mass spectrometry, and mitoplasting experiments .

• Copper Transport: Beyond simply chelating copper, CUP1 may function as a "depository" for copper designated for transfer into apo forms of copper proteins, suggesting a more active role in copper trafficking within cells .

• Interaction with Low-Molecular-Mass Complexes: Mitochondrial CUP1 may limit the concentrations of low-molecular-mass copper complexes in the organelle, potentially preventing toxic reactions or facilitating controlled copper distribution .

These discoveries suggest that CUP1 functions as a multifunctional protein involved in several stress response pathways and cellular compartments, making it an increasingly important target for comprehensive study in yeast cellular biology.

How can I design experiments to investigate the dual role of CUP1 in both copper and nitrosative stress responses?

To investigate the dual role of CUP1 in copper and nitrosative stress responses, consider these experimental approaches:

• Genetic Manipulation Strategy:

  • Generate strains with varying CUP1 expression levels: cup1Δ (deletion), wild-type, and CUP1 overexpression

  • Create CUP1 mutants with altered copper-binding capacity to separate copper and NO-related functions

  • Develop inducible CUP1 expression systems to control timing of expression relative to stress exposure

• Stress Response Characterization:

  • Sequential stress testing: Apply copper stress followed by nitrosative stress (or vice versa) at various intervals

  • Combined stress testing: Expose cells to both stressors simultaneously at different concentrations

  • Quantification methods: Measure growth rates, survival percentages, and metabolic activity

  • Molecular markers: Monitor oxidative/nitrosative damage to proteins, lipids, and DNA

• Protein Function Analysis:

  • NO scavenging assay: Use DAF-FM fluorescence to measure NO levels in various genetic backgrounds

  • Copper chelation assay: Quantify copper binding under different NO concentrations

  • Structural studies: Investigate if NO interaction alters CUP1's copper-binding properties

  • Post-translational modifications: Determine if nitrosation affects CUP1 function

• Subcellular Distribution Studies:

  • Fractionation experiments: Isolate cytosolic and mitochondrial fractions under different stress conditions

  • Live cell imaging: Use fluorescently tagged CUP1 to track relocalization during stress responses

  • Immuno-electron microscopy: Precisely locate CUP1 within mitochondrial subcompartments

  • Protein import assays: Test if stress affects CUP1 import into mitochondria

• Interactome Analysis:

  • Co-immunoprecipitation: Identify protein partners under copper stress, nitrosative stress, or both

  • Proximity labeling: Use BioID or APEX2 fused to CUP1 to catalog neighbors under different conditions

  • Genetic interaction screening: Perform synthetic genetic array analysis with cup1Δ under different stresses

These approaches will help elucidate the mechanisms by which CUP1 contributes to both copper homeostasis and nitrosative stress tolerance, potentially revealing new therapeutic targets for conditions involving metal toxicity and nitrosative stress.

What new antibody-based techniques are being developed for studying metallothioneins like CUP1?

Several innovative antibody-based techniques are emerging for studying metallothioneins like CUP1:

• Metal-Sensitive Antibodies:

  • New antibodies specifically designed to distinguish between metal-bound and metal-free forms of metallothioneins

  • These conformation-specific antibodies can track the metalation status of CUP1 in different cellular compartments

  • Applications include monitoring dynamic changes in CUP1-copper binding during stress responses

• Proximity Ligation Assays (PLA):

  • This technique detects protein-protein interactions with high sensitivity using paired antibodies

  • For CUP1 research, PLA can identify interactions between CUP1 and copper transport proteins

  • The method provides spatial information about where in the cell these interactions occur

  • Quantifiable signals allow measurement of interaction dynamics during stress responses

• Multiplexed Imaging Technologies:

  • Mass cytometry (CyTOF) using metal-tagged antibodies allows simultaneous detection of dozens of proteins

  • Imaging mass cytometry combines CyTOF with laser ablation for subcellular resolution

  • These approaches can reveal how CUP1 expression correlates with other stress response proteins at the single-cell level

  • Particularly useful for studying heterogeneous responses within yeast populations

• Nanobody Development:

  • Single-domain antibodies derived from camelid antibodies

  • Their small size allows access to epitopes that might be sterically hindered for conventional antibodies

  • Can be expressed intracellularly as "intrabodies" to track CUP1 in living cells

  • Potential for developing conformation-specific nanobodies that recognize specific metal-bound states of CUP1

• Quantitative Super-Resolution Microscopy:

  • Combining super-resolution techniques (STORM, PALM) with CUP1-specific antibodies

  • Enables visualization of CUP1 distribution at nanometer resolution

  • Can detect clustering and co-localization with other proteins at previously unresolvable scales

  • Particularly valuable for studying CUP1 distribution within mitochondrial subcompartments

These emerging techniques expand the toolkit available for metallothionein research, potentially providing new insights into the complex roles of CUP1 in cellular stress responses and copper homeostasis.