COPS8 Antibody

What is COPS8 and what cellular pathways does it participate in?

COPS8 (also known as CSN8 or SGN8) is one of the eight subunits of the COP9 signalosome (CSN), an evolutionarily conserved protein complex that functions as an important regulator in multiple signaling pathways. The structure and function of the COP9 signalosome is similar to that of the 19S regulatory particle of 26S proteasome . The CSN complex plays an essential role in the ubiquitin conjugation pathway by mediating the deneddylation of SCF-type E3 ligase complexes, which leads to decreased ubiquitin ligase activity . This process is critical for protein degradation and cellular homeostasis.

COPS8 is involved in phosphorylation of several proteins including p53, c-jun/JUN, ITPK1, and IRF8/ICSBP, likely through its association with CK2 and PKD kinases . Recent studies have demonstrated that COPS8 is essential for Drosophila development and plays a crucial role in peripheral T cell homeostasis and antigen receptor-induced entry into the cell cycle from quiescence . Additionally, COPS8 acts downstream of the AhR pathway and is required for protective effects in certain inflammatory conditions .

What are the key specifications of commercially available COPS8 antibodies?

Commercial COPS8 antibodies are available in various formats with distinct specifications based on host species, reactivity, and applications. Most commonly, these antibodies are raised in rabbits and demonstrate reactivity with human, mouse, and rat samples . The antibodies target COPS8, which has a calculated and observed molecular weight of approximately 23 kDa .

These antibodies have been validated in various cell lines and tissues including SH-SY5Y cells, HepG2 cells, T-47D cells, NIH/3T3 cells, mouse brain tissue, rat brain tissue, and human intrahepatic cholangiocarcinoma tissue .

What are the optimal conditions for Western blot detection of COPS8?

For optimal Western blot detection of COPS8, researchers should consider several key parameters including antibody dilution, sample preparation, and detection methods. Based on available data, COPS8 antibodies should be used at dilutions ranging from 1:5000-1:50000 or 1:200-1:2000 depending on the specific product .

The 23 kDa molecular weight of COPS8 means it runs relatively low on standard SDS-PAGE gels, so using an appropriate percentage gel (12-15%) is recommended for optimal resolution. When preparing samples from tissues, mouse brain tissue has demonstrated consistent positive results with COPS8 antibodies . Cell line samples that reliably express detectable COPS8 include SH-SY5Y cells, HepG2 cells, T-47D cells, and NIH/3T3 cells .

For challenging samples or weak signals, researchers should consider:

• Optimizing lysis buffer composition to ensure complete protein extraction

• Using fresh samples when possible

• Including protease inhibitors during sample preparation

• Extending primary antibody incubation time to overnight at 4°C

• Using enhanced chemiluminescence (ECL) detection systems with appropriate exposure times

What are the recommended protocols for immunohistochemical detection of COPS8?

For immunohistochemical (IHC) detection of COPS8, the recommended antibody dilutions range from 1:125-1:500 or 1:20-1:200 depending on the specific product and sample type . The following protocol elements are critical for successful COPS8 IHC:

Antigen Retrieval Methods:
COPS8 antibodies work optimally with specific antigen retrieval procedures. For human intrahepatic cholangiocarcinoma tissue, suggested antigen retrieval should be performed with TE buffer at pH 9.0, although citrate buffer at pH 6.0 can be used as an alternative . This step is crucial for exposing epitopes that may be masked during fixation.

Detection Systems:
Both DAB-based chromogenic detection and fluorescence-based systems have proven effective for COPS8 visualization in tissue sections. The choice depends on the researcher's need for colocalization studies, signal quantification, or morphological assessment.

Positive Control Tissues:
Mouse brain tissue and human gliomas tissue have been validated as positive controls for COPS8 IHC applications . Including appropriate positive controls is essential for confirming antibody performance.

How can I validate the specificity of COPS8 antibodies in my experimental system?

Validating antibody specificity is crucial for ensuring reliable research results. For COPS8 antibodies, several complementary approaches are recommended:

• Genetic Validation: Utilize COPS8 knockout or knockdown models as negative controls. For example, intestinal epithelium-specific COPS8 knockout mice (COPS8ΔIEC) have been generated and characterized . Comparing antibody signals between wild-type and knockout tissues provides definitive evidence of specificity.

• Peptide Competition Assays: Pre-incubate the COPS8 antibody with excess immunizing peptide (if available, such as the VGLPVEEAVK GILEQGWQAD STTRMVLPRK PVAGALDVSF NKFIPLSEPA P sequence) before application to samples. Disappearance of signal indicates specific binding.

• Multiple Antibody Validation: Use different antibodies targeting distinct epitopes of COPS8. Concordant results with antibodies from different sources increase confidence in specificity.

• Molecular Weight Verification: Confirm that the detected band corresponds to the expected molecular weight of COPS8 (23 kDa) . Multiple or unexpected bands may indicate cross-reactivity or protein degradation.

• Cross-Species Validation: COPS8 antibodies show reactivity with human, mouse, and rat samples . Highest antigen sequence identity to mouse is 88% and to rat is 90% . Consistent detection across species with expected expression patterns supports antibody specificity.

What is known about the role of COPS8 in gut inflammation and how can antibodies help study this function?

Recent research has identified critical roles for COPS8 in intestinal homeostasis and inflammation. Studies utilizing gut epithelium-specific knockout of COPS8 have demonstrated that COPS8 acts downstream of the AhR pathway and is required for protective effects against inflammation .

COPS8 deficiency leads to:

• Reduced expression of antimicrobial peptides (AMPs) such as Defensin Alpha-1, Defa-21, Defa-b1, Defa-rs1, and Angiopoietin-4 in the ileum and colon

• Altered gut microbiota composition, particularly increased Segmented Filamentous Bacteria (SFB)

• Increased susceptibility to intestinal inflammation with severe weight loss and mortality in experimental models

• Elevated pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) in the colon

Antibody applications for studying COPS8 in gut inflammation include:

• Immunohistochemistry: To assess COPS8 expression patterns in different intestinal cell types and changes during inflammation

• Western blotting: For quantitative evaluation of COPS8 protein levels in intestinal tissues under various conditions

• Immunofluorescence: To analyze colocalization with inflammatory markers or cell-type specific markers

• Immunoprecipitation: To investigate protein-protein interactions that may be altered during inflammatory conditions

How does COPS8 knockout affect the intestinal microbiome and what techniques can be used to study this relationship?

COPS8 knockout in intestinal epithelial cells leads to significant alterations in gut microbiota composition. Metagenomic analysis of gut microbiota from COPS8ΔIEC mice revealed:

• Similar abundance of Bacteroidetes in both COPS8ΔIEC and control mice

• Higher abundance of Gram-negative bacteria related to Proteobacteria in COPS8ΔIEC mice

• Lower abundance of Gram-positive bacteria related to Firmicutes in COPS8ΔIEC mice

• Beta-Proteobacteria identified as specific for the microbiota in COPS8ΔIEC mice through linear discriminant analysis effect size (LEfSe) analysis

• Relatively higher levels of Clostridiales (related to Gram-positive Clostridia) in control mice compared to COPS8ΔIEC mice

These alterations correlate with reduced antimicrobial peptide production by Paneth cells in COPS8-deficient mice. The abnormal microbiota composition likely contributes to the increased susceptibility to intestinal inflammation observed in these animals.

Techniques for studying the COPS8-microbiome relationship include:

• 16S rRNA gene-based microbiota sequencing for taxonomic profiling

• Real-time PCR with 16S rRNA primers targeting specific bacterial populations

• Transmission electron microscopy to visualize bacteria-epithelium interactions

• Quantitative analysis of mucus-associated versus luminal bacteria

• Correlation of microbiota changes with inflammatory markers and antimicrobial peptide expression levels

What controls should be included when using COPS8 antibodies for different applications?

Proper controls are essential for interpreting results obtained with COPS8 antibodies. For different applications, the following controls should be considered:

For Western Blotting:

• Positive Control: Include lysates from tissues or cell lines known to express COPS8, such as mouse brain tissue, SH-SY5Y cells, HepG2 cells, or T-47D cells

• Negative Control: Include samples from COPS8 knockout models or cell lines with COPS8 knockdown

• Loading Control: Use antibodies against housekeeping proteins (e.g., GAPDH, β-actin) to ensure equal loading across lanes

• Molecular Weight Marker: Include a marker covering the 20-25 kDa range to confirm the expected 23 kDa size of COPS8

• Primary Antibody Omission: Process one membrane without primary antibody to identify non-specific binding of secondary antibody

For Immunohistochemistry:

• Positive Control Tissue: Include sections of mouse brain tissue or human intrahepatic cholangiocarcinoma tissue

• Negative Control Tissue: Include tissue sections from COPS8 knockout animals or tissues known not to express COPS8

• Primary Antibody Omission: Process one section without primary antibody

• Isotype Control: Use non-specific rabbit IgG at the same concentration as the COPS8 antibody

• Peptide Competition: Pre-incubate antibody with immunizing peptide to confirm specificity

For Immunofluorescence:

• Positive Control Cells: Include SH-SY5Y cells, which have been validated for positive IF/ICC detection

• Counterstaining: Use DAPI or another nuclear stain to visualize cellular context

• Secondary Antibody Control: Omit primary antibody to assess background fluorescence

• Autofluorescence Control: Include unstained samples to identify potential tissue autofluorescence

How should I optimize COPS8 antibody protocols for different tissue and cell types?

Optimizing COPS8 antibody protocols requires systematic adjustment of several parameters based on the specific tissue or cell type being studied:

For Tissue Sections:

• Antigen Retrieval: Test both TE buffer (pH 9.0) and citrate buffer (pH 6.0) for optimal epitope exposure

• Antibody Dilution: Perform a dilution series using the recommended ranges (1:125-1:500 or 1:20-1:200 for IHC)

• Incubation Time: Compare standard (1-2 hours at room temperature) versus extended incubation (overnight at 4°C)

• Blocking: Evaluate different blocking reagents (BSA, normal serum, commercial blockers) for minimizing background

• Section Thickness: Optimize section thickness (typically 4-6 μm for paraffin sections) for ideal antibody penetration

For Cell Lines:

• Fixation Method: Compare different fixatives (4% paraformaldehyde, methanol, acetone) and fixation times

• Permeabilization: Test detergents at various concentrations (0.1-0.5% Triton X-100, 0.05-0.2% Tween-20) for optimal intracellular access

• Cell Density: Ensure appropriate cell density for clear visualization of cellular structures

• Antibody Concentration: Begin with the middle of the recommended dilution range and adjust as needed

• Detection System: Select appropriate secondary antibodies based on desired visualization method

For both tissues and cells, it is advisable to first validate the antibody on known positive controls (SH-SY5Y cells, mouse brain tissue, or human intrahepatic cholangiocarcinoma tissue) before proceeding to experimental samples.

How can COPS8 antibodies be used to study the relationship between COP9 signalosome function and disease pathogenesis?

COPS8 antibodies provide valuable tools for investigating the role of the COP9 signalosome in various pathological conditions. The COP9 signalosome regulates protein degradation pathways that are implicated in numerous diseases, including cancer, neurodegenerative disorders, and inflammatory conditions.

For inflammatory bowel disease research, COPS8 antibodies can be used to:

• Assess COPS8 expression changes in patient biopsies compared to healthy controls

• Evaluate effects of therapeutic interventions on COPS8 levels and localization

• Investigate interactions between COPS8 and components of the AhR pathway

• Monitor changes in COPS8 expression during progression of intestinal inflammation

• Study correlations between COPS8 levels and antimicrobial peptide production

In neurological research, COPS8 antibodies can be applied to:

• Examine COPS8 expression patterns in different regions of the brain

• Investigate potential alterations in neurodegenerative conditions

• Study COPS8 interactions with neuronal proteins involved in protein degradation pathways

• Assess changes in COPS8 localization during neuronal development or disease progression

For cancer research applications:

• Evaluate COPS8 expression in various tumor types including intrahepatic cholangiocarcinoma

• Investigate associations between COPS8 levels and tumor aggressiveness or treatment response

• Study potential interactions between COPS8 and cancer-related signaling pathways

• Explore COPS8 as a potential biomarker or therapeutic target

What approaches can be used to study interactions between COPS8 and other components of the COP9 signalosome?

Investigating protein-protein interactions involving COPS8 requires specialized techniques that can be enhanced with appropriate antibody applications:

• Co-immunoprecipitation (Co-IP): COPS8 antibodies can be used to pull down COPS8 and its interaction partners from cell or tissue lysates. This approach can identify both known and novel interactions with other COP9 signalosome components or substrates.

• Proximity Ligation Assay (PLA): This technique uses pairs of antibodies (including anti-COPS8) to detect proteins in close proximity (<40 nm), providing evidence of potential interactions in situ with subcellular resolution.

• Bimolecular Fluorescence Complementation (BiFC): While not directly using antibodies, this method can complement antibody-based approaches by visualizing protein interactions in living cells.

• Chromatin Immunoprecipitation (ChIP): COPS8 antibodies can be used to identify potential DNA-binding activities of COPS8-containing complexes, providing insights into transcriptional regulatory functions.

• Immunofluorescence Colocalization: COPS8 antibodies used in conjunction with antibodies against other signalosome components can demonstrate spatial colocalization at the subcellular level.

• Mass Spectrometry following Immunoprecipitation: COPS8 antibodies can be used to isolate protein complexes that can then be analyzed by mass spectrometry to identify interaction partners under different conditions.

• FRET-based Assays: When combined with fluorescently labeled secondary antibodies, these approaches can demonstrate proximity between COPS8 and other proteins of interest.