EXOC7 Antibody, HRP conjugated

What is EXOC7 and what cellular functions does it perform?

EXOC7, also known as Exocyst Complex Component 7 or Exo70, is a critical subunit of the exocyst complex. This protein plays essential roles in:

• Constitutive protein secretion of soluble proteins

• Post-Golgi trafficking to the plasma membrane

• Plasma membrane tethering of secretory vesicles

• Dendritic arbor formation and spine maturation in hippocampal neurons

• Modulation of protein degradation pathways through interaction with ubiquitin E3 ligases

Recent research defines the exocyst complex, including EXOC7, as the molecular tether for constitutive protein secretion and an essential component of the secretory pathway . The protein has a predicted molecular weight of 83 kDa based on its 735 amino acid sequence, though it typically appears at approximately 74-80 kDa on Western blots .

What are the standard applications for EXOC7 antibody, HRP conjugated?

EXOC7 antibody, HRP conjugated is primarily utilized in the following applications:

The HRP conjugation provides direct enzymatic detection capability, eliminating the need for secondary antibody incubation steps, which can be particularly advantageous for reducing background signal and streamlining experimental protocols .

What specific storage requirements are necessary for maintaining EXOC7 antibody, HRP conjugated activity?

For optimal preservation of activity, EXOC7 antibody, HRP conjugated should be stored according to these guidelines:

• Temperature: Store at -20°C or -80°C immediately upon receipt

• Avoid repeated freeze-thaw cycles, which can significantly reduce antibody activity

• For HRP-conjugated antibodies in particular, aliquoting is recommended to prevent activity loss from repeated freeze-thaw cycles

• Storage buffer typically contains 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as preservative

Note that appropriate storage is crucial for maintaining the HRP enzymatic activity, which can be more sensitive to degradation than the antibody protein itself .

How can I optimize Western blotting protocols specifically for EXOC7 detection using HRP-conjugated antibodies?

Optimizing Western blotting for EXOC7 detection requires attention to several technical considerations:

Sample Preparation:

• Include protease inhibitor cocktail (e.g., P8340, Sigma-Aldrich) when preparing cell lysates

• For ubiquitination studies, treat cells with 5 μM MG-132 (proteasome inhibitor) before harvesting

• Use appropriate lysis buffer: 10 mM HEPES pH 7.5, 5 mM MgCl₂, 142.5 mM KCl, 1 mM EDTA, 10% glycerol, and 1% Triton X-100 works well for EXOC7 extraction

Blotting Parameters:

• Expected molecular weight: 74-80 kDa

• Recommended dilution: 1:500-1:3000 for standard WB

• Positive controls: Human brain tissue, HEK-293 cells, and mouse brain tissue have shown reliable EXOC7 expression

Signal Development:

• With HRP-conjugated antibodies, use enhanced chemiluminescence (ECL) substrate directly

• Shorter exposure times (30-60 seconds) may be sufficient to prevent oversaturation

• For low abundance samples, consider using high-sensitivity ECL substrates

Validation Strategy:

• Always include positive controls (HEK-293 cells or human brain tissue)

• Consider knockdown controls using EXOC7-specific siRNA/shRNA to confirm specificity

What is the functional relationship between EXOC7 and protein degradation pathways in neurodegenerative disorders?

EXOC7 (Exo70) plays a complex role in protein degradation pathways, particularly in the context of polyglutamine diseases:

• Opposing Function to Prpf19: EXOC7/exo70 modulates expanded ATXN3-polyQ protein levels and toxicity in an opposite manner to Prpf19/prp19 (an E3 ubiquitin ligase)

• Regulatory Mechanism: EXOC7 exerts its ATXN3-polyQ-modifying effect through regulating the E3 ligase function of Prpf19/prp19

• Balance in Protein Degradation: A fine balance between Prpf19 and EXOC7 is critical for achieving degradation of disease proteins in spinocerebellar ataxia type 3 (SCA3)

• Experimental Evidence: Studies in both mammalian and Drosophila disease models have demonstrated that manipulation of EXOC7 levels affects the accumulation of polyglutamine-expanded proteins

This relationship suggests potential therapeutic targets for neurodegenerative disorders characterized by protein aggregation. Researchers investigating this pathway typically employ co-immunoprecipitation assays to detect EXOC7-Prpf19 interactions and in vitro ubiquitination assays to assess effects on protein degradation .

How can I design effective co-immunoprecipitation experiments to study EXOC7 interactions?

Designing effective co-immunoprecipitation (co-IP) experiments for EXOC7 requires careful consideration of multiple technical factors:

Optimized Protocol:

• Seed HEK293 cells in six-well plates and transfect for 48 hours

• Wash cells twice with ice-cold 1× PBS

• Add binding buffer (10 mM HEPES, pH 7.5, 5 mM MgCl₂, 142.5 mM KCl, 1 mM EDTA, 10% glycerol, and 1% Triton X-100) supplemented with protease inhibitor cocktail

• Sonicate samples (duty cycle 30%, output control 3, 30 seconds)

• Incubate at 4°C for 1 hour with gentle rotation

• Centrifuge at 4°C for 20 minutes at 14,000 × g

• Save a fraction of supernatant as "Input"

• Wash Dynabeads protein G with binding buffer three times

• Add pre-cleared beads and antibodies to remaining samples

• Incubate at 4°C for 16 hours with gentle rotation

• Include a beads-only control (no primary antibody)

• Wash beads three times with binding buffer and resuspend in SDS sample buffer

• Heat all protein samples at 99°C for 10 minutes prior to immunoblotting analysis

Known Interaction Partners:

• Prpf19/prp19 (E3 ubiquitin ligase)

• RAL GTPases (RALA/RALB) - function in exocyst complex regulation

• Other exocyst components (EXOC1, EXOC3, EXOC5, EXOC6)

Technical Considerations:

• Use appropriate negative controls (beads only, IgG control)

• For detecting transient interactions, consider using crosslinking agents

• For studying membrane-associated interactions, optimize detergent conditions

What role does EXOC7 play in the secretory pathway and post-Golgi trafficking?

EXOC7 serves critical functions in the secretory pathway and post-Golgi trafficking:

• Essential Tethering Component: The exocyst complex, including EXOC7, functions as the molecular tether for constitutive protein secretion of soluble proteins

• Post-Golgi Carrier Association: EXOC7 localizes to post-Golgi carriers and is enriched at fusion hotspots on the plasma membrane

• Functional Requirement: CRISPR-knockout studies demonstrate that the exocyst complex is essential for the arrival of post-Golgi carriers to the plasma membrane

• Molecular Association: EXOC7 specifically colocalizes with LAMP1Δ-RUSH carriers near the plasma membrane, as demonstrated by TIRF imaging

• Biochemical Evidence: EXOC7 (along with other exocyst subunits) is enriched in immunoprecipitated LAMP1Δ-RUSH post-Golgi carriers

• Regulatory Mechanisms: The function of EXOC7 in secretory trafficking is regulated by RAL GTPases (RALA/RALB) and phosphoinositide signaling through PIP5K enzymes

This essential role is demonstrated through carrierIP assays showing enrichment of EXOC7 in post-Golgi carriers, and through functional assays showing defects in protein secretion following EXOC7 depletion .

What approaches can be used to validate the specificity of EXOC7 antibodies in experimental systems?

Validating antibody specificity is critical for reliable research. For EXOC7 antibodies, consider these validation approaches:

Genetic Validation:

• Transient CRISPR-knockout of EXOC7 followed by Western blotting to confirm loss of specific band

• siRNA/shRNA-mediated knockdown to demonstrate reduced signal intensity correlating with reduced protein expression

• Overexpression of tagged EXOC7 to confirm co-localization with antibody signal

Biochemical Validation:

• Immunoprecipitation followed by mass spectrometry to confirm identity of pulled-down protein

• Testing across multiple cell lines/tissues with known EXOC7 expression patterns

• Comparison with multiple antibodies targeting different epitopes of EXOC7

Application-Specific Controls:

• For WB: Include positive control samples (human brain tissue, HEK-293 cells, mouse brain tissue)

• For IHC: Include isotype control antibodies and blocking peptide competition

• For IF/ICC: Perform peptide competition assays and co-staining with other exocyst markers

Cross-Reactivity Assessment:

• Test antibody against recombinant EXOC7 fragments to confirm epitope specificity

• Evaluate potential cross-reactivity with related exocyst components

Using these validation approaches ensures reliable detection of EXOC7 and increases confidence in experimental results, particularly when employing HRP-conjugated antibodies that may exhibit different specificity profiles than unconjugated versions.

How does the HRP conjugation affect the applications and performance of EXOC7 antibodies?

HRP (horseradish peroxidase) conjugation provides distinct advantages and considerations for EXOC7 antibodies:

Advantages:

• Direct detection capability eliminates secondary antibody requirements, reducing protocol time and potential cross-reactivity issues

• Lower background in applications like ELISA and IHC due to elimination of secondary antibody binding to endogenous immunoglobulins

• Quantitative signal correlation with target concentration, making it suitable for quantitative assays

• Compatibility with various substrates (TMB, DAB, ECL) for flexible detection options

Performance Considerations:

• Slightly higher detection limit compared to amplification methods using secondary antibodies

• Empirical dilution optimization required (typically starting at 1:1000 for WB applications)

• HRP enzymatic activity may be more sensitive to storage conditions than the antibody itself

• Avoiding sodium azide in buffers is critical as it inhibits HRP activity

Application-Specific Performance:

• ELISA: Primary validated application with excellent sensitivity

• WB: Effective but may require optimization of exposure times to prevent oversaturation

• IHC: Suitable but requires special consideration for antigen retrieval (TE buffer pH 9.0 recommended)

What troubleshooting approaches are effective when EXOC7 antibody detection yields unexpected results?

When facing challenges with EXOC7 antibody detection, consider these systematic troubleshooting approaches:

No Signal or Weak Signal:

• Verify sample preparation - ensure protease inhibitors were included

• Increase antibody concentration (try 1:500 dilution for WB)

• Extend primary antibody incubation time (overnight at 4°C)

• For HRP-conjugated antibodies, ensure fresh substrate and absence of sodium azide

• Verify target expression in your sample (EXOC7 is expressed in human brain tissue, HEK-293 cells)

Multiple Bands/Non-specific Binding:

• Increase blocking stringency (5% BSA or milk in TBST)

• Add 0.1-0.5% Tween-20 to washing buffer

• Reduce antibody concentration (try 1:3000 dilution for WB)

• Extend washing steps (5 × 5 minutes)

• Consider the presence of isoforms or post-translational modifications of EXOC7

Inconsistent Results:

• Standardize sample preparation protocols

• Ensure consistent incubation times and temperatures

• Prepare fresh working solutions of antibody dilutions

• For HRP-conjugated antibodies, avoid repeated freeze-thaw cycles

• Use positive control samples across experiments (HEK-293 cells)

High Background:

• Increase blocking time and concentration

• Reduce antibody concentration

• For HRP-conjugated antibodies, reduce substrate incubation time

• Use fresh reagents and buffers

• For tissue sections, increase washing steps and consider adding 0.3% H₂O₂ treatment to quench endogenous peroxidases

What experimental design considerations are important when studying EXOC7 in protein trafficking and secretion?

When designing experiments to investigate EXOC7's role in protein trafficking and secretion, consider these critical parameters:

Cell Models:

• HEK293 cells provide a reliable model system for EXOC7 studies

• Primary hippocampal neurons are valuable for studying EXOC7's neuronal functions

• Cell-type specific differences in exocyst complex composition should be considered

Trafficking Assays:

• RUSH (retention using selective hooks) system provides quantitative trafficking data

• Design synthetic reporter proteins (e.g., LAMP1Δ-RUSH) for visualizing post-Golgi carriers

• Combine with TIRF microscopy to visualize fusion events at the plasma membrane

Genetic Manipulation:

• Transient CRISPR-knockout system is effective for studying exocyst components

• Consider redundancy between related proteins (e.g., RAL GTPases have overlapping functions)

• Rescue experiments with tagged EXOC7 constructs can confirm specificity of phenotypes

Interaction Studies:

• CarrierIP assay effectively isolates post-Golgi carriers for protein interaction studies

• Co-immunoprecipitation with optimized binding buffers captures EXOC7 complexes

• Consider membrane association when designing lysis and immunoprecipitation protocols

Functional Readouts:

• Cell surface ratio quantification (FACS) provides quantitative measures of trafficking efficiency

• Secretomics approaches identify broad effects on protein secretion

• Combine with imaging approaches to correlate biochemical data with spatial information

Implementing these experimental design considerations ensures robust investigation of EXOC7's functions in cellular trafficking pathways.

How is EXOC7 implicated in neurodegenerative disease mechanisms?

EXOC7 has emerging roles in neurodegenerative disease mechanisms through several pathways:

Protein Degradation Regulation:

• EXOC7 modulates the activity of Prpf19, an E3 ubiquitin ligase involved in protein degradation

• This regulatory relationship affects the accumulation of polyglutamine-expanded proteins in spinocerebellar ataxia type 3 (SCA3)

• Imbalance between EXOC7 and Prpf19 may contribute to the pathological accumulation of misfolded proteins

Neuronal Morphology and Function:

• EXOC7 modulates dendrite arbor formation, synapse density, and spine maturation in primary hippocampal neurons

• Disruptions in these processes are common features across multiple neurodegenerative disorders

Secretory Pathway Regulation:

• As an essential component of the secretory pathway, EXOC7 dysfunction could impact the secretion of neurotrophic factors and other proteins crucial for neuronal health

• Impaired protein trafficking is increasingly recognized as a contributory factor in neurodegeneration

Potential Therapeutic Implications:

• Modulating the balance between EXOC7 and protein degradation machinery represents a potential therapeutic strategy

• Understanding EXOC7's interactions may reveal novel drug targets for diseases characterized by protein aggregation

These findings suggest that EXOC7-targeting strategies may have therapeutic potential in neurodegenerative disorders where protein homeostasis is disrupted.

What experimental approaches can determine EXOC7's spatial and temporal dynamics during vesicle fusion events?

Investigating EXOC7's spatial and temporal dynamics during vesicle fusion requires sophisticated imaging and biochemical approaches:

Advanced Imaging Techniques:

• Total Internal Reflection Fluorescence (TIRF) microscopy captures EXOC7 localization at the plasma membrane interface during fusion events

• Live-cell imaging with fluorescently tagged EXOC7 (e.g., HALO-EXOC6) reveals dynamic association with vesicle carriers

• Super-resolution microscopy (STED, PALM, STORM) can resolve nanoscale organization of EXOC7 at fusion sites

Reporter Systems:

• RUSH (retention using selective hooks) system enables synchronized release of cargo from the Golgi for temporal analysis

• Dual-color imaging with differentially labeled EXOC7 and vesicle markers establishes temporal relationship during tethering and fusion

• pH-sensitive fluorescent cargo reporters can precisely time fusion pore opening in relation to EXOC7 recruitment

Biochemical Approaches:

• CarrierIP assay isolates post-Golgi carriers at different time points to monitor temporal changes in EXOC7 association

• Proximity labeling approaches (BioID, APEX) can identify proteins near EXOC7 during different trafficking stages

• Optogenetic manipulation of EXOC7 recruitment allows direct testing of its temporal requirements

Correlative Techniques:

• Correlative light and electron microscopy (CLEM) connects EXOC7 dynamics with ultrastructural features of vesicle tethering

• Fluorescence Recovery After Photobleaching (FRAP) quantifies EXOC7 turnover rates at fusion sites

These approaches collectively provide a comprehensive view of EXOC7's dynamic behavior during vesicle trafficking and fusion events.

What are the best experimental approaches for studying EXOC7's role in ubiquitination pathways?

Studying EXOC7's role in ubiquitination pathways requires specialized techniques:

In Vitro Ubiquitination Assays:

• Treat HEK293 cells with 5 μM MG-132 (proteasome inhibitor) before harvesting

• Lyse cells in ubiquitination lysis buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2% SDS) with protease inhibitor cocktail

• Heat homogenate at 99°C for 10 minutes followed by sonication

• Add ubiquitination dilution buffer and rotate at 4°C for 1 hour

• Save a portion as "input" and proceed with immunoprecipitation

• Use stringent washing conditions (ubiquitination washing buffer: 10 mM Tris-HCl, pH 8.0, 1 M NaCl, 1 mM EDTA, and 1% NP-40)

• Analyze by immunoblotting for ubiquitinated species

Interaction Studies:

• Co-immunoprecipitation assays to detect EXOC7-Prpf19 interactions under different cellular conditions

• Proximity ligation assays (PLA) to visualize EXOC7-E3 ligase interactions in situ

• Domain mapping experiments to identify interaction interfaces between EXOC7 and ubiquitination machinery

Functional Approaches:

• Expression of EXOC7 variants (e.g., CC domain only, NES-EXOC7) to determine domains required for ubiquitination regulation

• Cellular ubiquitination assays using HA-tagged ubiquitin to monitor global effects of EXOC7 manipulation

• Pulse-chase experiments to measure protein degradation rates when EXOC7 levels or activity are modulated

Genetic Models:

• CRISPR/Cas9 knockout or knockdown of EXOC7 followed by proteomics to identify changes in the ubiquitinated proteome

• Drosophila and mammalian disease models to assess in vivo effects of EXOC7 manipulation on protein degradation