COPS7B Antibody

What is COPS7B and why is it important in cellular research?

COPS7B is a subunit of the COP9 signalosome complex that plays a crucial role in regulating protein degradation pathways. This complex is essential for cell cycle control, signal transduction, and DNA repair mechanisms . Research has shown that COPS7B is particularly important because:

• It contributes to the assembly and function of the COP9 signalosome complex

• Dysregulation of COPS7B has been implicated in various diseases, including cancer

• It serves as a promising target for therapeutic interventions

• It has regulatory roles in cellular circadian function

The significance of COPS7B extends beyond basic cellular processes, as it has been identified in disease mechanisms including IMiD resistance in multiple myeloma and as part of a translational regulation axis in colorectal cancer .

How do COPS7B antibodies differ from other COP9 signalosome component antibodies?

COPS7B antibodies specifically target the COPS7B subunit rather than other components of the COP9 signalosome complex. When designing experiments, researchers should consider:

• Antibody specificity: COPS7B antibodies are designed to recognize epitopes unique to COPS7B, such as specific amino acid sequences (e.g., AA 61-95 or AA 1-157)

• Cross-reactivity: While some antibodies may show reactivity with both human and mouse COPS7B, others may be species-specific

• Applications: Different COPS7B antibodies may be optimized for specific applications (WB, IHC, IF, ELISA) with varying recommended dilutions

Unlike antibodies targeting other COP9 signalosome components, COPS7B antibodies enable specific investigation of this subunit's unique functions, such as its role in CRBN stability for IMiD efficacy and in regulating translational efficiency in cancer cells .

What expression patterns and subcellular localization does COPS7B exhibit in normal tissues?

COPS7B exhibits specific expression patterns important for experimental interpretation:

• Temporal expression: COPS7B transcripts show markedly circadian patterns of expression, suggesting time-dependent regulatory functions

• Nuclear import: The COP9 signalosome complex containing COPS7B is imported into the nucleus in a timed fashion to stabilize the essential circadian protein BMAL1

• Tissue variability: COPS7B is expressed in various tissues, with detected expression in brain and stomach tissues in mouse models

When designing immunofluorescence experiments, researchers should account for this temporal and spatial variability by:

• Collecting samples at consistent time points when studying circadian effects

• Using appropriate nuclear and cytoplasmic markers to precisely track localization

• Comparing expression levels across multiple tissue types to establish baseline expression

What are the optimal protocols for using COPS7B antibodies in Western blotting?

Western blotting with COPS7B antibodies requires specific optimization:

For optimal results:

• Include positive controls from tissues known to express COPS7B (e.g., brain tissue)

• Perform membrane blocking with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Incubate with primary antibody overnight at 4°C for maximum sensitivity

• Wash thoroughly (4-5 times) with TBST before and after secondary antibody incubation

• Validate specificity by comparing observed band sizes to predicted molecular weights (30 kDa)

How can I optimize immunohistochemistry protocols for COPS7B detection in tissue samples?

Immunohistochemistry (IHC) for COPS7B requires specific considerations:

• Sample preparation:

  • Use either paraffin-embedded or frozen sections based on your specific requirements

  • For FFPE tissues, perform antigen retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

• Antibody conditions:

  • Use recommended dilutions (typically 1:20-1:200)

  • Incubate at 4°C overnight for optimal sensitivity

  • Secondary antibody selection should match the host species (typically rabbit)

• Signal detection and validation:

  • Use DAB or AEC as chromogen depending on your experimental needs

  • Include known positive controls (colorectal cancer tissues show increased COPS7B)

  • Perform parallel negative controls (omitting primary antibody)

  • Consider dual IHC with other COP9 signalosome components to validate specificity

• Quantification approach:

  • Score staining intensity on a 0-3 scale

  • Assess percentage of positive cells

  • Calculate H-score (intensity × percentage) for semi-quantitative analysis

What methods enable accurate quantification of COPS7B levels in experimental samples?

Multiple methods can provide quantitative assessment of COPS7B:

Western Blot Densitometry:

• Capture images using a digital imaging system with linear dynamic range

• Normalize COPS7B band intensity to loading controls (β-actin, GAPDH)

• Use standard curves with recombinant COPS7B protein for absolute quantification

ELISA-Based Quantification:

• Use COPS7B antibodies at recommended dilutions (1:2000-1:10000)

• Generate standard curves with recombinant COPS7B protein

• Include technical replicates (minimum 3) and biological replicates

Flow Cytometry:

• Permeabilize cells for intracellular COPS7B detection

• Use antibody concentrations optimized for flow cytometry

• Quantify using median fluorescence intensity

• Include isotype controls to determine background staining

RT-qPCR for Transcriptional Analysis:

• Design primers specific to COPS7B transcript variants

• Normalize to appropriate reference genes

• Correlate transcript levels with protein expression to identify post-transcriptional regulation

How does COPS7B contribute to IMiD resistance in multiple myeloma?

COPS7B plays a critical role in immunomodulatory drug (IMiD) resistance mechanisms in multiple myeloma:

• Genomic basis for resistance:

  • Chromosome 2q37, which contains COPS7B and COPS8, shows progressive copy loss in IMiD-resistant myeloma

  • Copy loss increases from newly diagnosed (~5%) to lenalidomide-resistant (10%) and pomalidomide-resistant (16.4%) cases

  • The proportion of cases where copy loss becomes clonal increases with disease progression

• Molecular mechanism:

  • COPS7B is required for CRBN stability, the target protein of IMiD drugs

  • Partial loss of COPS7B leads to partial loss of CRBN, which reduces efficacy of IMiD drugs

  • Patients with COPS7B/COPS8 loss show significantly poorer progression-free survival when treated with pomalidomide

• Experimental considerations:

  • Researchers should assess copy number alterations of COPS7B when studying IMiD resistance

  • Genomic changes at 2q37 can be monitored during treatment to predict therapeutic response

  • Combined assessment of COPS7B and CRBN levels provides more comprehensive understanding of resistance mechanisms

What is the role of COPS7B in translational regulation and cancer progression?

COPS7B has emerging functions in translational regulation with implications for cancer:

• Translational efficiency (TE) in colorectal cancer:

  • COPS7B shows significantly increased translational efficiency in colorectal cancer without transcriptional alteration

  • It functions as part of the "ribo-interactome" that interacts with ribosomes

  • The IGF2BP3–COPS7B axis facilitates ribosome biogenesis and mRNA translation initiation

• Mechanistic basis:

  • IGF2BP3 enhances the translational efficiency of COPS7B mRNA

  • Increased COPS7B expression elevates translational efficiency of genes involved in protein synthesis

  • This creates a positive feedback loop accelerating cancer growth and metastasis

• Experimental approaches:

  • Ribosome profiling can be used to measure translational efficiency of COPS7B

  • RNA immunoprecipitation can detect IGF2BP3 binding to COPS7B mRNA

  • Polysome profiling can assess COPS7B association with active ribosomes

This represents a novel function distinct from COPS7B's role in the COP9 signalosome, highlighting the importance of studying context-dependent functions.

How does COPS7B influence circadian rhythms at the molecular level?

COPS7B has unexpected roles in circadian regulation:

• Genetic variation and circadian phenotypes:

  • Genome-wide association studies have linked COPS7B genetic variants to cellular circadian function

  • These variations contribute to differences in human chronotypes ("larks" vs. "owls")

• Molecular mechanisms:

  • COPS7B transcripts exhibit markedly circadian expression patterns

  • The COP9 signalosome is imported into the nucleus in timed fashion

  • COPS7B-containing complexes stabilize the essential circadian protein BMAL1

  • This represents a novel mechanism opposing BMAL1's proteasome-mediated degradation

• Experimental considerations:

  • Circadian studies involving COPS7B should control for time of sample collection

  • Both genotype and COPS7B expression levels should be considered when analyzing circadian phenotypes

  • Studies should examine nuclear-cytoplasmic shuttling of COPS7B throughout the circadian cycle

These findings suggest that circadian clock properties depend partially on genetically-encoded competition between stabilizing forces (including COPS7B) and destabilizing forces.

What are common pitfalls in COPS7B antibody-based experiments and how can they be addressed?

Researchers should be aware of several technical challenges:

• Non-specific binding:

  • Problem: Multiple bands in Western blots beyond expected 30 kDa size

  • Solution: Increase blocking time/concentration, optimize antibody dilution, validate with recombinant COPS7B protein

• Temporal variation effects:

  • Problem: Inconsistent results due to circadian expression of COPS7B

  • Solution: Standardize sample collection times, record and report collection time in publications

• Cross-reactivity with COPS7A:

  • Problem: Potential cross-reactivity with the paralogous COPS7A protein

  • Solution: Use COPS7B antibodies targeting unique epitopes; consider knockout/knockdown controls to validate specificity

• Epitope masking:

  • Problem: False negatives due to protein interactions or post-translational modifications

  • Solution: Try multiple antibodies targeting different COPS7B epitopes; use denaturing conditions for Western blots

• Variability between antibody lots:

  • Problem: Different lots of the same antibody may show performance variations

  • Solution: Validate each new lot against previous results; maintain detailed records of antibody performance

How can I validate the specificity of COPS7B antibodies for my experimental system?

Rigorous validation approaches include:

• Genetic validation:

  • CRISPR/Cas9 knockout of COPS7B as negative control

  • siRNA/shRNA knockdown showing corresponding reduction in signal

  • Overexpression systems showing increased signal intensity

• Peptide competition assays:

  • Pre-incubate antibody with immunizing peptide (e.g., COPS7B AA 61-95)

  • Compare signal with and without peptide competition

  • Specific binding should be blocked by immunizing peptide

• Multiple antibody validation:

  • Test multiple antibodies against different COPS7B epitopes

  • Consistent results across antibodies increase confidence in specificity

  • Compare polyclonal and monoclonal antibodies when available

• Mass spectrometry verification:

  • Immunoprecipitate with COPS7B antibody

  • Confirm identity of pulled-down proteins by mass spectrometry

  • Verify presence of COPS7B and expected interacting proteins

• Cross-species reactivity testing:

  • Test antibody in species with high and low sequence homology

  • Correlate signal with known evolutionary conservation patterns

  • Verify reactivity matches manufacturer's claims (e.g., human, mouse)

What advanced techniques can be combined with COPS7B antibodies for comprehensive functional analysis?

Integrating multiple techniques provides deeper insights:

• Proximity ligation assays (PLA):

  • Detect protein-protein interactions between COPS7B and other COP9 signalosome components

  • Visualize interactions with CRBN in the context of IMiD resistance

  • Quantify associations with ribosomal proteins in translational regulation studies

• ChIP-seq combined with COPS7B immunoprecipitation:

  • Identify chromatin regions associated with COPS7B

  • Map temporal binding patterns in circadian regulation studies

  • Connect COPS7B to transcriptional regulation mechanisms

• CRISPR screens with COPS7B antibody validation:

  • Perform genome-wide CRISPR screens for COPS7B function

  • Validate phenotypes using COPS7B antibodies in orthogonal assays

  • Identify synthetic lethal interactions in cancer contexts

• Live-cell imaging with fluorescently tagged antibody fragments:

  • Track COPS7B dynamics in real-time using cell-permeable antibody fragments

  • Correlate localization with cell cycle phases

  • Observe nuclear-cytoplasmic shuttling during circadian cycles

• Spatial transcriptomics with COPS7B immunostaining:

  • Correlate COPS7B protein levels with local transcriptomic signatures

  • Identify spatial domains of COPS7B activity in tissue contexts

  • Connect protein localization to function in complex tissues