COPS3 Antibody

What is COPS3 and what is its biological significance?

COPS3 (COP9 Signalosome Complex Subunit 3), also known as CSN3 or SGN3, is a critical component of the COP9 signalosome complex (CSN) that regulates various cellular and developmental processes. This protein functions as an essential regulator of the ubiquitin conjugation pathway by mediating the deneddylation of cullin subunits of SCF-type E3 ligase complexes . This activity decreases the ubiquitin ligase activity of complexes such as SCF, CSA, or DDB2, playing a vital role in protein degradation pathways. Additionally, the complex participates in the phosphorylation of several proteins including p53/TP53, c-jun/JUN, IkappaBalpha/NFKBIA, ITPK1, and IRF8/ICSBP, potentially through its association with CK2 and PKD kinases . The CSN-dependent phosphorylation differentially affects protein fate; for instance, it promotes TP53 degradation while protecting JUN from degradation via the ubiquitin system.

What are the molecular characteristics of the COPS3 protein?

COPS3 is a 48 kDa protein (calculated molecular weight) that is widely expressed across tissues, with particularly high expression in heart and skeletal muscle . The protein sequence contains specific domains that facilitate its interaction with other components of the COP9 signalosome. The human COPS3 protein sequence includes crucial regions that serve as epitopes for antibody recognition, with many commercial antibodies targeting the C-terminal region (approximately amino acids 300-400) . Understanding these molecular characteristics is essential for selecting appropriate antibodies and designing experiments to study COPS3 function.

What types of COPS3 antibodies are available for research applications?

COPS3 antibodies are available in several formats with varying characteristics that make them suitable for different research applications:

These antibodies provide researchers with options for different experimental needs, including detection of endogenous COPS3 levels and specific epitopes within the protein .

How should I select a COPS3 antibody for my specific research application?

When selecting a COPS3 antibody, consider these factors based on your experimental goals:

• Application compatibility: Ensure the antibody is validated for your intended application (WB, IHC, IF, etc.) . Some antibodies perform better in specific applications due to differences in epitope accessibility under various experimental conditions.

• Species reactivity: Verify that the antibody reacts with your model organism. Many COPS3 antibodies react with human, mouse, and rat proteins, while some have predicted reactivity with additional species like pig, bovine, horse, and others .

• Epitope location: Select antibodies targeting specific regions if you're investigating particular domains or if post-translational modifications might mask certain epitopes. C-terminal antibodies are common for COPS3 .

• Clonality: Choose between polyclonal antibodies (broader epitope recognition, higher sensitivity) and monoclonal antibodies (higher specificity, better reproducibility) based on your experimental needs .

• Validation data: Review the manufacturer's validation data, including Western blot images showing the expected 48 kDa band and immunohistochemistry results demonstrating appropriate cellular localization .

If your research involves studying protein interactions within the COP9 signalosome complex, consider antibodies that target regions not involved in these interactions to avoid epitope masking effects.

What are the optimal conditions for Western blot detection of COPS3?

For optimal Western blot detection of COPS3, follow these methodological guidelines:

• Sample preparation: Prepare whole cell lysates using RIPA buffer supplemented with protease inhibitors. For HeLa or 293T cells, load approximately 50 μg of total protein per lane .

• Protein separation: Use 10-12% SDS-PAGE gels to achieve optimal resolution around the 48 kDa region where COPS3 migrates.

• Transfer conditions: Transfer proteins to PVDF membranes using standard protocols (typically 100V for 60-90 minutes in Tris-glycine buffer with 20% methanol).

• Blocking: Block membranes with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature.

• Primary antibody incubation: Dilute COPS3 antibodies according to manufacturer recommendations. For example, ab12321 can be used at 0.04 μg/mL , while other antibodies may require different concentrations. Incubate overnight at 4°C.

• Detection system: Use appropriate HRP-conjugated secondary antibodies and enhanced chemiluminescence (ECL) detection. Short exposure times (e.g., 10 seconds) may be sufficient for detecting COPS3 .

• Expected results: A single band at approximately 48 kDa should be visible, representing the COPS3 protein . If examining over-expressed COPS3, include both mock-transfected and COPS3-transfected samples as controls .

How should I optimize immunohistochemistry protocols for COPS3 detection?

For successful immunohistochemical detection of COPS3 in tissue samples:

• Tissue preparation: Use formalin-fixed, paraffin-embedded (FFPE) tissues sectioned at 4-5 μm thickness . For frozen sections, fix briefly in 4% paraformaldehyde before proceeding.

• Antigen retrieval: Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) for 15-20 minutes. This critical step unmasks epitopes that may be cross-linked during fixation.

• Blocking: Block endogenous peroxidase with 3% hydrogen peroxide, followed by blocking non-specific binding with 5-10% normal serum from the same species as the secondary antibody.

• Primary antibody: Dilute COPS3 antibody appropriately. For example, ab12321 has been successfully used at 1/1000 (1μg/ml) for human prostate carcinoma tissue . Incubate overnight at 4°C or for 1-2 hours at room temperature.

• Detection system: Use a polymer-based detection system compatible with your primary antibody species (e.g., DAB for visualization) .

• Counterstaining: Counterstain with hematoxylin to visualize cell nuclei, then dehydrate and mount sections.

• Controls: Always include positive controls (tissues known to express COPS3, such as heart or skeletal muscle) and negative controls (primary antibody omitted) to validate staining specificity.

What procedures should I follow for immunofluorescence detection of COPS3?

For immunofluorescence detection of COPS3 in cultured cells:

• Cell preparation: Culture cells on coverslips or chamber slides to 70-80% confluence.

• Fixation: Fix cells with 4% paraformaldehyde for 15 minutes at room temperature, then permeabilize with 0.1-0.5% Triton X-100 for 10 minutes.

• Blocking: Block with 5% normal serum from the secondary antibody species in PBS containing 0.1% Triton X-100 for 1 hour at room temperature.

• Primary antibody: Apply diluted COPS3 antibody (follow manufacturer's recommendations for IF applications) and incubate overnight at 4°C in a humidified chamber.

• Secondary antibody: Incubate with appropriate fluorophore-conjugated secondary antibody (e.g., Alexa Fluor 488 or 594) for 1 hour at room temperature in the dark.

• Nuclear counterstain: Counterstain with DAPI or Hoechst to visualize nuclei.

• Mounting: Mount coverslips using anti-fade mounting medium to preserve fluorescence.

• Expected localization: COPS3 typically shows both nuclear and cytoplasmic localization, as the COP9 signalosome complex functions in both compartments. Verify this pattern when interpreting results.

How can I utilize COPS3 antibodies for co-immunoprecipitation studies?

Co-immunoprecipitation (Co-IP) with COPS3 antibodies enables investigation of protein-protein interactions within the COP9 signalosome complex:

• Antibody selection: Choose COPS3 antibodies specifically validated for immunoprecipitation (IP) . Ensure the antibody targets an epitope that doesn't interfere with protein-protein interactions of interest.

• Cell lysis: Prepare cell lysates using a gentle non-denaturing lysis buffer (e.g., 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40) supplemented with protease and phosphatase inhibitors.

• Pre-clearing: Pre-clear lysates with Protein A/G beads to reduce non-specific binding.

• Immunoprecipitation: Incubate pre-cleared lysates with COPS3 antibody (2-5 μg per 1 mg of total protein) overnight at 4°C with gentle rotation. Add fresh Protein A/G beads and continue incubation for 2-4 hours.

• Washing: Wash immunoprecipitates 4-5 times with lysis buffer to remove non-specifically bound proteins.

• Elution and analysis: Elute bound proteins by boiling in SDS-PAGE sample buffer, then analyze by Western blotting using antibodies against potential interacting partners (e.g., other COP9 signalosome components, cullins, or substrate proteins).

• Controls: Always include a negative control using non-specific IgG from the same species as the COPS3 antibody to establish background binding levels.

When investigating COPS3 interactions with cullin proteins or other signalosome components, gentle lysis conditions are crucial to preserve native protein complexes.

What are common troubleshooting strategies for non-specific binding with COPS3 antibodies?

When encountering non-specific binding with COPS3 antibodies:

• Optimize antibody concentration: Titrate the antibody to determine the optimal concentration that maximizes specific signal while minimizing background. For Western blots, test dilutions ranging from 1:500 to 1:5000.

• Adjust blocking conditions: Increase blocking time or try different blocking agents (BSA vs. non-fat dry milk). For some applications, 5% BSA may provide better blocking than milk proteins.

• Modify washing steps: Increase the number and duration of washing steps. Adding 0.1-0.5% Tween-20 to wash buffers can help reduce non-specific interactions.

• Verify antibody specificity:

  • Use peptide competition assays where the antibody is pre-incubated with the immunizing peptide, which should eliminate specific binding .

  • Include positive controls (cell lines known to express COPS3) and negative controls (COPS3 knockdown cells).

• Sample preparation considerations: Ensure complete protein denaturation for Western blotting by adequate heating in sample buffer. For IP applications, optimize lysis conditions to maintain protein conformation while reducing non-specific interactions.

• Secondary antibody checks: Test secondary antibody alone to rule out non-specific binding from this reagent.

If multiple bands appear in Western blots, consider that they may represent isoforms, degradation products, or post-translationally modified versions of COPS3 rather than non-specific binding.

How can I quantify COPS3 expression levels in experimental and control samples?

For accurate quantification of COPS3 expression:

• Western blot quantification:

  • Use housekeeping proteins (β-actin, GAPDH, α-tubulin) as loading controls.

  • Capture images within the linear range of detection to ensure accurate densitometry.

  • Analyze band intensity using software such as ImageJ, normalizing COPS3 signal to the loading control.

  • Run a dilution series of a positive control sample to establish a standard curve.

• Immunohistochemistry quantification:

  • Use digital image analysis software to quantify DAB staining intensity.

  • Score staining based on intensity (0-3+) and percentage of positive cells.

  • Apply the H-score method: H-score = Σ(i × Pi), where i = intensity (0-3) and Pi = percentage of cells with that intensity.

  • Include calibration standards in each batch of staining to account for inter-batch variability.

• Flow cytometry quantification:

  • Use COPS3 antibodies validated for flow cytometry .

  • Include isotype controls to establish background staining levels.

  • Use fluorescence minus one (FMO) controls to set accurate gates.

  • Report data as median fluorescence intensity (MFI) or percentage of positive cells.

• qRT-PCR correlation:

  • Compare protein expression data with mRNA levels to corroborate findings.

  • Use specific primers targeting the COPS3 gene for accurate transcript quantification.

For all quantification methods, perform technical replicates (minimum of three) and biological replicates to ensure statistical validity of the results.

How can COPS3 antibodies be used to investigate the role of COPS3 in disease mechanisms?

COPS3 antibodies can be powerful tools for exploring disease mechanisms:

• Cancer research applications:

  • Evaluate COPS3 expression in normal versus tumor tissues using immunohistochemistry with antibodies like ab12321 .

  • Correlate COPS3 expression with clinical parameters and patient outcomes.

  • Investigate how COPS3 affects the stability of tumor suppressors or oncoproteins via the COP9 signalosome's deneddylation activity.

• Neurodegenerative disease studies:

  • Examine COPS3 levels in brain tissues from patients with neurodegenerative disorders.

  • Investigate COPS3's role in protein aggregate clearance through its function in the ubiquitin-proteasome system.

• Developmental biology:

  • Use immunofluorescence with COPS3 antibodies to track protein expression during developmental processes .

  • Combine with lineage markers to understand tissue-specific roles.

• Drug development applications:

  • Use COPS3 antibodies to screen for compounds that modulate COP9 signalosome activity.

  • Monitor changes in COPS3 levels or localization in response to therapeutic interventions.

• Mechanistic studies:

  • Combine COPS3 antibodies with proximity ligation assays to visualize protein interactions in situ.

  • Use chromatin immunoprecipitation (ChIP) to investigate potential roles in transcriptional regulation.

The adaptability of COPS3 antibodies across multiple techniques makes them valuable for translational research connecting molecular mechanisms to disease phenotypes.

What techniques can be used to study COPS3 post-translational modifications?

To investigate post-translational modifications (PTMs) of COPS3:

• Phosphorylation analysis:

  • Use phospho-specific antibodies if available for known COPS3 phosphorylation sites.

  • Perform immunoprecipitation with general COPS3 antibodies followed by Western blotting with anti-phosphoserine/threonine/tyrosine antibodies.

  • Combine with phosphatase treatment as a control to confirm phosphorylation status.

• Mass spectrometry approaches:

  • Immunoprecipitate COPS3 using validated antibodies .

  • Analyze the purified protein by LC-MS/MS to identify PTMs.

  • Use SILAC (Stable Isotope Labeling with Amino acids in Cell culture) to quantify changes in PTMs under different conditions.

• 2D gel electrophoresis:

  • Separate proteins first by isoelectric point and then by molecular weight.

  • Detect COPS3 isoforms using specific antibodies in Western blotting.

  • Changes in the spot pattern may indicate alterations in PTMs.

• In vitro kinase assays:

  • Immunoprecipitate COPS3 and incubate with purified kinases.

  • Detect phosphorylation using radioactive ATP incorporation or phospho-specific antibodies.

• Mobility shift assays:

  • Observe changes in COPS3 migration patterns on SDS-PAGE following treatment with phosphatase inhibitors or activators of specific kinase pathways.

  • Confirm shifts using Phos-tag™ SDS-PAGE, which specifically retards the migration of phosphorylated proteins.

These approaches can reveal how PTMs regulate COPS3 function within the COP9 signalosome complex and its interactions with other proteins.

How can I design experiments to study the dynamics of COPS3 in the COP9 signalosome assembly?

To investigate COPS3 dynamics in COP9 signalosome assembly:

• Fluorescence Recovery After Photobleaching (FRAP):

  • Generate cells expressing COPS3-GFP fusion proteins.

  • Photobleach a region of interest and monitor fluorescence recovery over time.

  • Calculate diffusion coefficients and binding/unbinding rates to assess COPS3 mobility.

• Bimolecular Fluorescence Complementation (BiFC):

  • Fuse complementary fragments of a fluorescent protein to COPS3 and other CSN subunits.

  • When proteins interact, the fragments reconstitute an active fluorophore, enabling visualization of complex formation in living cells.

• Proximity-dependent labeling:

  • Generate COPS3 fusion proteins with BioID or APEX2 enzymes.

  • Identify proteins in close proximity to COPS3 during CSN assembly through biotinylation and subsequent purification.

• Size exclusion chromatography:

  • Fractionate cell lysates to separate protein complexes based on size.

  • Use COPS3 antibodies in Western blotting to detect which fractions contain COPS3.

  • Compare fractionation patterns under different conditions (e.g., cell cycle stages, stress).

• Cross-linking mass spectrometry:

  • Treat cells with protein cross-linkers to stabilize transient interactions.

  • Immunoprecipitate COPS3-containing complexes.

  • Analyze by mass spectrometry to identify cross-linked peptides, revealing spatial relationships within the complex.

• Single-molecule tracking:

  • Label COPS3 with photo-convertible fluorescent proteins or quantum dots.

  • Track individual molecules in living cells to determine dynamics at the single-molecule level.

These techniques can provide insights into how COPS3 incorporates into the COP9 signalosome and how this process is regulated in different cellular contexts or disease states.

What are the recommended validation steps for COPS3 antibodies in research?

Comprehensive validation of COPS3 antibodies should include:

• Specificity testing:

  • Peptide competition assays using the immunizing peptide .

  • Testing in COPS3 knockout/knockdown samples versus wild-type.

  • Comparing results across multiple COPS3 antibodies targeting different epitopes.

• Application-specific validation:

  • For Western blotting: Verify single band at expected molecular weight (48 kDa) .

  • For IHC/IF: Compare staining patterns with published literature and verify subcellular localization is consistent with known COPS3 biology.

  • For IP: Confirm enrichment of COPS3 in immunoprecipitated samples versus input.

• Cross-reactivity assessment:

  • Test antibodies against recombinant COPS3 protein.

  • Evaluate potential cross-reactivity with other COP9 signalosome components due to structural similarities.

• Reproducibility verification:

  • Perform technical replicates to ensure consistent results.

  • Document lot-to-lot variation when using different antibody batches.

• Publication standards:

  • Record complete antibody information including catalog number, lot number, dilution, and incubation conditions.

  • Include validation data in publications or supplementary materials.

Following these validation steps ensures reliable and reproducible results when using COPS3 antibodies for research applications.

What emerging technologies might enhance COPS3 research in the future?

Several emerging technologies hold promise for advancing COPS3 research:

• CRISPR-based techniques:

  • CRISPR knock-in of fluorescent tags or epitope tags to endogenous COPS3 for live-cell imaging and improved antibody detection.

  • CRISPRi/CRISPRa for precise control of COPS3 expression levels.

  • Base editing or prime editing for introducing specific mutations to study structure-function relationships.

• Advanced imaging approaches:

  • Super-resolution microscopy (STORM, PALM, SIM) to visualize COPS3 within the COP9 signalosome at nanometer resolution.

  • Light-sheet microscopy for high-speed 3D imaging of COPS3 dynamics in living cells or tissues.

  • Lattice light-sheet microscopy for reduced phototoxicity during long-term imaging.

• Single-cell technologies:

  • Single-cell proteomics to examine COPS3 levels and modifications in individual cells.

  • Spatial transcriptomics combined with COPS3 antibody staining to correlate protein localization with gene expression patterns.

• Protein structure determination:

  • Cryo-EM for high-resolution structures of the COP9 signalosome with COPS3 in different functional states.

  • AlphaFold2 and other AI-based protein structure prediction tools to model COPS3 interactions.

• Multiplexed protein detection:

  • CycIF (cyclic immunofluorescence) or CODEX for simultaneous detection of COPS3 and dozens of other proteins in the same sample.

  • Mass cytometry (CyTOF) incorporating COPS3 antibodies for high-dimensional analysis of signaling networks.