GOPC Antibody

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

GOPC (Golgi-associated PDZ and coiled-coil motif-containing protein) is a multifunctional protein that plays critical roles in intracellular protein trafficking and degradation pathways. It has been implicated in regulating CFTR chloride currents and acid-induced ASIC3 currents by modulating cell surface expression of both channels. Additionally, GOPC may regulate the intracellular trafficking of the ADR1B receptor and plays a role in autophagy. Together with MARCHF2, it mediates the ubiquitination and lysosomal degradation of CFTR. Its overexpression results in CFTR intracellular retention and lysosomal degradation, making it an important target for research in cellular biology and protein trafficking studies .

What are the different names and identifiers associated with GOPC?

GOPC is known by several alternative names in scientific literature, which can sometimes cause confusion when searching for relevant antibodies. The table below summarizes the various identifiers and aliases:

Understanding these alternative nomenclatures is essential when reviewing literature and selecting appropriate antibodies for your research .

Where is GOPC protein localized in cells and tissues?

GOPC exhibits specific subcellular localization patterns that are important to consider when designing immunostaining experiments. It is primarily found in the cytoplasm and associated with the Golgi apparatus membrane as a peripheral membrane protein. It is also present in the trans-Golgi network membrane. In neuronal cells, GOPC is enriched in synaptosomal and postsynaptic densities (PSD) fractions, cell junctions, synapses, and dendrites. In Purkinje cells, it is expressed in cell bodies and dendrites. In reproductive tissues, it is localized at the trans-Golgi network of spermatids and the medulla of round spermatides. This diverse localization pattern reflects its multiple roles in cellular trafficking and protein processing .

How do I select the appropriate GOPC antibody for my specific research application?

Selecting the right GOPC antibody requires careful consideration of several factors. First, determine your application requirements (WB, IHC, ELISA, etc.) and ensure the antibody has been validated for that specific application. Check the reactivity to confirm it recognizes your species of interest (human, mouse, etc.). Review the immunogen information to understand what region of GOPC the antibody targets - antibodies targeting different epitopes may have varying performance in different applications. For instance, the C-terminal antibody targets the region between 357-386 amino acids of human GOPC .

Also consider the clonality (polyclonal vs. monoclonal) - polyclonals offer broader epitope recognition but potentially more background, while monoclonals provide higher specificity. Finally, look for validation data in applications similar to yours, and when possible, select antibodies that have been cited in peer-reviewed publications for your specific application .

What validation methods should I use to confirm the specificity of a GOPC antibody?

Validation is critical for ensuring reproducible results with GOPC antibodies. A comprehensive validation approach should include:

• Positive and negative controls: Test the antibody on tissues or cell lines known to express GOPC (positive) and those that don't (negative). This is considered a fundamental validation approach by manufacturers .

• Western blot analysis: Confirm the antibody detects a band of the expected molecular weight (approximately 50.52 kDa for GOPC).

• Knockout/knockdown validation: Compare antibody staining in wild-type versus GOPC knockout or knockdown samples.

• Overexpression studies: Test in systems where GOPC is overexpressed to confirm increased signal.

• Peptide competition assay: Pre-incubate the antibody with its immunogen peptide to confirm signal reduction.

• Cross-reactivity testing: Evaluate if the antibody cross-reacts with related proteins.

For advanced validation, consider using orthogonal methods to confirm findings, such as mass spectrometry or RNA sequencing to correlate protein expression with antibody signal 5.

How does antibody format and storage affect GOPC antibody performance?

The format and storage conditions significantly impact antibody performance and longevity. Most GOPC antibodies are supplied as purified polyclonal antibodies in PBS with additives like sodium azide (0.09-0.02%) and glycerol (often 50%). The purification process typically involves protein A column purification, sometimes followed by peptide affinity purification .

For storage, manufacturers consistently recommend keeping GOPC antibodies refrigerated at 2-8°C for short-term storage (up to 2 weeks) and at -20°C for long-term storage. To prevent degradation from freeze-thaw cycles, it's advisable to store the antibody in small aliquots. Some suppliers recommend -80°C for extended storage periods .

Improper storage can lead to antibody degradation, resulting in diminished specificity, increased background, and inconsistent staining patterns. Always check the certificate of analysis for specific storage recommendations for your particular antibody .

What are the optimal protocols and dilutions for using GOPC antibodies in Western blotting?

For Western blotting applications with GOPC antibodies, optimal results can be achieved by following these methodological guidelines:

• Sample preparation: Prepare cell or tissue lysates in a compatible lysis buffer containing protease inhibitors to prevent protein degradation.

• Protein loading: Load 20-40 μg of total protein per lane, depending on GOPC expression levels in your sample.

• Antibody dilutions: Based on manufacturer recommendations, the optimal dilution ranges are:

  • For antibody source : 1:1000

  • For antibody source : 1:500-1:5000

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

• Primary antibody incubation: Incubate membranes with diluted GOPC antibody overnight at 4°C.

• Detection method: Use compatible secondary antibodies and detection systems appropriate for your imaging equipment.

• Expected results: A predominant band should be visible at approximately 50.52 kDa, which corresponds to the calculated molecular weight of GOPC .

Optimize these conditions for your specific experimental system, particularly if you're working with samples that might have post-translational modifications or different isoforms of GOPC.

How can I optimize immunohistochemistry protocols for GOPC detection in tissue sections?

Optimizing immunohistochemistry (IHC-P) for GOPC detection requires attention to several critical parameters:

• Tissue fixation: Use 10% neutral buffered formalin for 24-48 hours, as overfixation can mask epitopes and reduce antibody binding.

• Antigen retrieval: Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 8.0) is recommended. Test both methods to determine which works best with your specific GOPC antibody.

• Blocking: Block endogenous peroxidase activity with 3% H₂O₂, followed by protein blocking with 5-10% normal serum.

• Antibody dilutions: Optimal dilutions vary by manufacturer:

  • For antibody source : 1:10-1:50

  • For antibody source : 1:20-1:200

• Incubation conditions: Incubate primary antibody overnight at 4°C in a humidified chamber.

• Detection system: Use a polymer or ABC detection system with DAB or AEC chromogen.

• Counterstaining: Hematoxylin counterstaining provides good nuclear contrast.

• Controls: Always include positive control tissues known to express GOPC (e.g., tissues with well-established Golgi apparatus structures) and negative controls (primary antibody omission) .

Start with the manufacturer's recommended dilution and adjust as needed based on your results. Signal intensity should be balanced against background staining.

What considerations are important when using GOPC antibodies for studying protein-protein interactions?

When investigating protein-protein interactions involving GOPC, several methodological considerations should be addressed:

• Antibody epitope selection: Choose antibodies that target regions of GOPC unlikely to be involved in the protein-protein interaction you're studying. The C-terminal region (amino acids 357-386) is commonly used for antibody generation , but this may interfere with interactions that involve the PDZ domain.

• Co-immunoprecipitation optimization: For co-IP experiments, use mild lysis buffers that preserve protein complexes (avoid harsh detergents). Consider crosslinking approaches for transient interactions.

• Technical controls: Include IgG controls, input controls, and when possible, GOPC knockout or knockdown controls to verify specificity.

• Proximity ligation assays (PLA): For in situ detection of GOPC interactions, PLA can provide spatial resolution of interactions within cellular compartments.

• Reciprocal co-IP: Confirm interactions by performing reciprocal co-IPs using antibodies against the interacting partner.

• Known interactions: Consider GOPC's established interactions with CFTR, MARCHF2, and ADR1B receptor as positive controls for your experimental system .

• Cellular compartment considerations: Since GOPC localizes to specific subcellular regions (Golgi, synapses, etc.), subcellular fractionation may improve detection of compartment-specific interactions .

Remember that antibody-based detection of protein complexes can sometimes disrupt the very interactions you're trying to study, so complementary approaches like recombinant protein pull-downs may be necessary.

What are common challenges when using GOPC antibodies and how can they be resolved?

Researchers often encounter several challenges when working with GOPC antibodies, each with specific troubleshooting approaches:

• High background signal:

  • Increase blocking time or concentration (5-10% serum/BSA)

  • Optimize antibody dilution (try more dilute solutions)

  • Include 0.1-0.3% Triton X-100 in blocking and antibody solutions for better penetration

  • Reduce primary antibody incubation time or temperature

• Weak or no signal:

  • Optimize antigen retrieval methods (try different buffers and times)

  • Decrease antibody dilution

  • Extend incubation time or increase temperature

  • Check if the epitope is masked by fixation (try different fixation methods)

  • Verify GOPC expression in your sample type

• Non-specific bands in Western blot:

  • Increase blocking time and/or concentration

  • Use more stringent washing conditions

  • Optimize primary antibody dilution

  • Consider using a different GOPC antibody targeting another epitope

• Inconsistent results between experiments:

  • Standardize all protocols and reagent preparations

  • Use the same lot of antibody when possible

  • Prepare larger volumes of working solutions to use across experiments

  • Store antibody aliquots properly to avoid freeze-thaw cycles 5

For advanced applications, consider using recombinant antibodies which tend to offer greater batch-to-batch consistency compared to traditional polyclonal antibodies, as mentioned in the webinar on antibodies and research reproducibility5.

How do I interpret contradictory results between different GOPC antibodies?

Contradictory results between different GOPC antibodies are not uncommon and require careful interpretation through a systematic approach:

• Epitope differences: Different antibodies target different regions of GOPC. Compare the immunogen information to understand what epitopes are being recognized. Antibodies targeting the C-terminal region (aa 357-386) may give different results from those targeting other domains .

• Validation status: Assess the level of validation for each antibody. Has it been validated in knockout/knockdown systems? Are there published studies using these antibodies in your specific application?

• Isoform specificity: Check if the antibodies recognize different GOPC isoforms. GOPC has multiple protein accessions (NP_001017408.1, NP_065132.1), which might represent different isoforms that are differentially detected .

• Technical validation: Perform side-by-side testing of antibodies using positive and negative controls. Include tissues or cell lines with confirmed GOPC expression patterns .

• Orthogonal methods: Validate findings using non-antibody methods such as mass spectrometry or mRNA expression analysis to determine which antibody results correlate better with independent measures of GOPC.

• Cross-reactivity: Consider potential cross-reactivity with related proteins, especially for polyclonal antibodies.

When publishing, clearly report which antibody was used (including catalog number and lot), and consider acknowledging limitations if contradictory results were observed with different antibodies 5.

How can I optimize conditions for detecting low abundance GOPC in specific cell types?

Detecting low-abundance GOPC requires specialized techniques and optimization strategies:

• Signal amplification methods:

  • Use tyramide signal amplification (TSA) systems that can increase sensitivity by 10-100 fold

  • Consider biotin-streptavidin amplification systems

  • For fluorescence applications, use high quantum yield fluorophores and sensitive detection systems

• Sample enrichment:

  • Perform subcellular fractionation to concentrate GOPC-containing compartments (Golgi, synaptosomal fractions, etc.)

  • Use immunoprecipitation to concentrate GOPC before detection

  • Consider proximity labeling methods for enhancing detection of low-abundance interactors

• Antibody selection and optimization:

  • Choose high-affinity antibodies with demonstrated sensitivity

  • Optimize antibody concentration through titration experiments

  • Extend primary antibody incubation times (overnight at 4°C or even up to 48 hours)

  • Consider using cocktails of multiple GOPC antibodies targeting different epitopes

• Reduce background interference:

  • Use highly specific blocking agents (like 10% serum from the same species as the secondary antibody)

  • Include additional blocking steps (e.g., avidin/biotin blocking for biotin-based systems)

  • Perform more extensive washing steps between antibody incubations

• Technical modifications:

  • For IHC, extend DAB development time while monitoring background

  • For fluorescence, use longer exposure times and z-stack imaging with deconvolution

  • Consider using specialized microscopy techniques like total internal reflection fluorescence (TIRF) for surface GOPC or super-resolution microscopy

How can GOPC antibodies be used to study its role in protein trafficking and degradation pathways?

GOPC antibodies can be powerful tools for investigating protein trafficking and degradation pathways through several advanced approaches:

• Co-localization studies: Use dual immunofluorescence with markers of different cellular compartments (Golgi, endosomes, lysosomes, etc.) to track GOPC's distribution and potential role in trafficking. Confocal microscopy with z-stack acquisition can provide spatial resolution of GOPC's association with vesicular compartments.

• Pulse-chase experiments: Combine antibody staining with pulse-chase approaches to track the movement of cargo proteins (like CFTR) and assess how GOPC affects their trafficking kinetics and degradation rates.

• Live cell imaging: For dynamic studies, consider using GOPC antibody fragments (Fab) conjugated to fluorophores for live cell applications, or use GOPC-GFP constructs validated against antibody staining patterns.

• Proteasomal versus lysosomal degradation: Use GOPC antibodies in combination with inhibitors of proteasomal (MG132) or lysosomal (bafilomycin A1) degradation to distinguish between these pathways and GOPC's role in each.

• Ubiquitination studies: Combine GOPC immunoprecipitation with ubiquitin western blotting to study its role in the ubiquitination of target proteins, particularly in the context of MARCHF2-mediated ubiquitination of CFTR .

• Autophagy connections: GOPC is implicated in autophagy pathways, so combining GOPC antibodies with markers of autophagy (LC3, p62) can reveal its role in this critical cellular process .

These approaches can provide mechanistic insights into how GOPC regulates the trafficking and degradation of membrane proteins like CFTR, which has important implications for diseases like cystic fibrosis.

What are the latest advances in antibody technology that can enhance GOPC research?

Recent technological advances have significantly expanded the capabilities of antibody-based GOPC research:

• Recombinant antibody technology: Unlike traditional antibodies derived from immunized animals, recombinant antibodies are produced through molecular cloning and expression systems. These offer superior batch-to-batch consistency and can be engineered for specific properties. For GOPC research, this means more reliable and reproducible results across experiments and between laboratories5.

• Nanobodies and single-domain antibodies: These smaller antibody fragments offer better penetration into tissues and cellular compartments, potentially improving detection of GOPC in structurally complex locations like the Golgi apparatus or post-synaptic densities.

• Proximity labeling approaches: Techniques like BioID or APEX2 can be combined with GOPC antibodies to identify proteins in close proximity to GOPC within living cells, providing insights into its interaction network.

• Multiplexed imaging: New multiplexed immunofluorescence techniques allow simultaneous detection of GOPC alongside numerous other proteins, enabling complex pathway analysis in single tissue sections.

• Open science antibody validation initiatives: Organizations like YCharOS are creating open access antibody characterization data, which can help researchers select higher quality GOPC antibodies and establish validation standards5.

• Automated validation platforms: High-throughput antibody validation systems are emerging that can test antibodies across multiple applications and conditions, providing more comprehensive performance data.

These advances address the reproducibility challenges in antibody research discussed in the recorded webinar "Antibodies and Research Reproducibility," which emphasized the importance of validated reagents and transparent methods in improving research quality5.

How can I design experiments to study the interaction between GOPC and CFTR in cystic fibrosis research?

Designing robust experiments to investigate GOPC-CFTR interactions in cystic fibrosis research requires careful planning and multiple complementary approaches:

• Cellular models: Use both overexpression systems and endogenous detection in relevant cell types:

  • Bronchial epithelial cells (e.g., CFBE41o- cells)

  • Pancreatic duct cells (e.g., PANC-1)

  • Intestinal cell lines (e.g., Caco-2)

• Co-immunoprecipitation strategy:

  • Perform bidirectional co-IPs (pull down GOPC and probe for CFTR, and vice versa)

  • Include negative controls (IgG, irrelevant proteins) and positive controls (known GOPC or CFTR interactors)

  • Use mild detergents (0.5-1% NP-40 or Digitonin) to preserve protein-protein interactions

  • Consider crosslinking approaches for transient interactions

• Microscopy approaches:

  • Super-resolution microscopy to visualize co-localization beyond the diffraction limit

  • FRET or FLIM-FRET to demonstrate direct protein-protein interactions

  • Proximity ligation assay (PLA) to visualize interactions in situ with single-molecule sensitivity

• Functional assays:

  • Measure CFTR chloride channel activity using patch-clamp or fluorescent indicators while manipulating GOPC levels

  • Track CFTR surface expression and internalization rates in the presence/absence of GOPC

  • Assess CFTR half-life and degradation pathways when GOPC is overexpressed or knocked down

• Domainspecific interactions:

  • Design experiments with truncated or mutated versions of both proteins to map interaction domains

  • Focus on the PDZ domain of GOPC and the C-terminal PDZ-binding motif of CFTR

  • Use peptide competition assays with synthetic CFTR C-terminal peptides

• Tripartite complex studies:

  • Investigate the GOPC-MARCHF2-CFTR complex formation and its role in CFTR ubiquitination

  • Use sequential immunoprecipitation to isolate the complete complex

These approaches can provide mechanistic insights into how GOPC regulates CFTR trafficking and degradation, potentially identifying new therapeutic targets for cystic fibrosis.

How are GOPC antibodies being used in neuroscience research?

GOPC antibodies are emerging as valuable tools in neuroscience research due to GOPC's enrichment in synaptosomal and postsynaptic density fractions. Several cutting-edge applications include:

• Synapse formation and plasticity: GOPC antibodies can be used to study its localization at synapses and potential role in trafficking synaptic proteins. Its presence in dendrites and cell bodies of Purkinje cells suggests specialized functions in these neuronal compartments.

• Neurodevelopmental studies: Tracking GOPC expression and localization during neuronal development can provide insights into its role in synaptogenesis and circuit formation.

• Neurological disorder models: Examining GOPC expression and localization in models of neurological disorders, particularly those involving protein trafficking defects or synaptic dysfunction.

• Multiple labeling approaches: Combining GOPC antibodies with markers for specific neuronal populations or subcellular structures to map its distribution across different brain regions and cell types.

• Activity-dependent changes: Studying whether neuronal activity alters GOPC localization or function, potentially linking protein trafficking mechanisms to synaptic plasticity.

When designing such experiments, researchers should consider the specific subcellular localization patterns of GOPC in neuronal cells, including its enrichment in postsynaptic densities and dendrites, particularly in Purkinje cells .

What are the methodological considerations for studying GOPC in cancer research?

GOPC (originally identified as "Fused in glioblastoma" or FIG) has important implications for cancer research that require specific methodological approaches:

• Tissue-specific expression analysis:

  • Use tissue microarrays with GOPC antibodies to analyze expression across multiple tumor types and compare with matched normal tissues

  • Consider dual staining with proliferation markers (Ki-67) or cancer stem cell markers to correlate GOPC expression with tumor cell populations

• Fusion protein detection:

  • Design specialized antibody approaches to detect GOPC fusion proteins (like GOPC-ROS1) that occur in certain cancers

  • Use antibodies targeting different domains to distinguish full-length GOPC from fusion variants

• Trafficking pathway analysis in cancer cells:

  • Investigate how GOPC affects trafficking of receptor tyrosine kinases or other signaling proteins frequently dysregulated in cancer

  • Study co-localization with endocytic and recycling markers to assess its impact on receptor turnover

• Functional cancer studies:

  • Perform GOPC knockdown/overexpression in cancer cell lines followed by proliferation, migration, and invasion assays

  • Correlate GOPC levels with response to targeted therapies, particularly those affecting receptor trafficking

• In vivo cancer models:

  • Use GOPC antibodies for immunohistochemical analysis of xenograft or genetic cancer models

  • Consider how tumor microenvironment factors may affect GOPC expression or function

• Patient sample considerations:

  • Optimize antigen retrieval for FFPE patient samples, which may require more aggressive conditions than cell lines

  • Validate antibodies specifically in the cancer types being studied, as protein modifications or isoform expression may vary

These approaches can help elucidate GOPC's role in cancer development and progression, potentially identifying new therapeutic targets or biomarkers.

How can computational approaches enhance antibody design and selection for GOPC research?

Computational approaches are revolutionizing antibody design and selection processes for GOPC research through several innovative methods:

• In silico epitope prediction: Advanced algorithms can analyze the GOPC protein sequence to identify optimal epitopes for antibody generation based on:

  • Surface accessibility and hydrophilicity

  • Sequence conservation across species (for cross-reactive antibodies)

  • Minimal overlap with functional domains if studying protein interactions

  • Uniqueness compared to related proteins to minimize cross-reactivity

• Structural biology integration: Computational modeling of GOPC structure can guide antibody design by:

  • Identifying conformational epitopes not apparent from sequence alone

  • Predicting how antibody binding might affect GOPC function

  • Selecting epitopes that remain accessible in native protein complexes

• Machine learning for antibody specificity:

  • Recent advances in inferring antibody specificity from experimental data can help design antibodies with customized binding profiles

  • These approaches can predict cross-reactivity and optimize specificity before experimental validation

• Database integration for validation planning:

  • Systems like SciCrunch help track antibody usage across scientific literature

  • Computational analysis of published results can identify antibodies with consistent performance records5

• High-throughput screening analysis:

  • Computational tools can analyze results from phage display or other high-throughput screening approaches to identify optimal antibody candidates

  • Design of experiments (DoE) approaches can reduce the number of experiments needed for comprehensive validation

These computational approaches, when combined with experimental validation, can significantly improve the quality and reproducibility of GOPC antibodies used in research, addressing many of the challenges discussed in the "Antibodies and Research Reproducibility" webinar5.