EXOC8 Antibody

What is EXOC8 and what is its role in cellular processes?

EXOC8, also known as Exocyst complex 84 kDa subunit or EXO84, is a component of the evolutionarily conserved exocyst complex. This multiprotein complex plays a crucial role in vesicular trafficking and the secretory pathway by targeting post-Golgi vesicles to the plasma membrane. EXOC8 specifically functions in the docking of exocytic vesicles with fusion sites on the plasma membrane and is a target of activated Ral subfamily of GTPases .

The exocyst complex, containing EXOC8, contributes to multiple cellular processes including:

• Ciliogenesis

• Cytokinesis

• Cell migration

• Autophagy

• Fusion of secretory vesicles

Recent research has revealed EXOC8's significance in neurodevelopment, with loss-of-function variants being associated with abnormalities in cerebral cortical development . This protein has a calculated molecular weight of 82 kDa and consists of 725 amino acids .

What are the optimal applications for different EXOC8 antibody types?

Different EXOC8 antibodies demonstrate varying efficacy across research applications, necessitating strategic selection based on experimental design:

For immunoblotting applications, an optimal concentration range of 0.04-0.4 μg/mL has been validated . In tissue-specific applications, antibody performance varies significantly across tissues, with greater sensitivity typically observed in tissues with higher EXOC8 expression levels such as brain and testis .

How should researchers distinguish between EXOC8 and EXOSC8 antibodies in experimental design?

Despite similar nomenclature, EXOC8 and EXOSC8 target fundamentally different proteins with distinct cellular functions:

EXOC8 (Exocyst Complex Component 8):

• 82 kDa molecular weight

• Component of the exocyst complex

• Functions in vesicular trafficking and membrane fusion

• Observed at 82 kDa by Western blot

EXOSC8 (Exosome Component 8):

• 30 kDa molecular weight

• Component of the exosome multi-enzyme ribonuclease complex

• Functions in mRNA degradation and processing

• Observed at 30-35 kDa by Western blot

When designing experiments, researchers must verify:

• Correct gene symbol (EXOC8 vs. EXOSC8)

• Appropriate molecular weight markers

• Expected cellular localization patterns

• Specific reactivity profiles for the selected antibody

Antibody documentation should explicitly state whether it targets EXOC8 (Gene ID: 149371) or EXOSC8 (Gene ID: 11340) .

What validation strategies should be employed to confirm EXOC8 antibody specificity?

Comprehensive validation of EXOC8 antibodies requires multiple complementary approaches:

Basic Validation Approaches:

• Western blot analysis at appropriate dilutions (1:1000-1:6000) to confirm the expected 82 kDa band

• Positive control selection from validated reactive tissues (e.g., human brain, testis)

• Immunofluorescence patterns consistent with exocyst complex localization

Advanced Validation Methods:

• Recombinant expression validation comparing signals from EXOC8-expressing and control cells

• Protein array testing against 364 human recombinant protein fragments to assess cross-reactivity

• Knockdown/knockout approaches using EXOC8-targeted siRNA or CRISPR-Cas9 systems to confirm signal specificity

• Tissue arrays spanning 44 normal human tissues and 20 common cancer types to establish tissue-specific expression patterns

Prestige Antibodies® undergo particularly rigorous validation protocols including enhanced validation through recombinant expression systems that demonstrate signal specificity .

How does EXOC8 dysfunction contribute to neurodevelopmental disorders?

Recent genetic studies have established EXOC8 as a critical factor in neurodevelopment, with loss-of-function variants linked to specific neurological conditions:

Neurodevelopmental Disorder With Microcephaly, Seizures, and Brain Atrophy:

• Characterized by intellectual impairment

• Associated with hypertonia and brisk reflexes

• MRI findings demonstrate brain atrophy

A consanguineous family study identified a novel homozygous nonsense mutation [EXOC8; NM_175876.5; c.1714G > T; p.(Glu572Ter)] in affected individuals. Both parents were heterozygous carriers of this mutation. This study confirmed that truncated EXOC8 protein leads to improper assembly of the exocyst complex, resulting in the accumulation of neurotransmitters and other excretory vesicles within cells .

The identification of this protein-truncating variant provides important insights for:

• Establishing genotype-phenotype correlations

• Genetic counseling approaches

• Clinical management strategies for affected individuals

What are the optimal sample preparation methods for EXOC8 immunodetection?

Sample preparation significantly impacts EXOC8 antibody performance across different applications:

For Western Blotting:

• Optimal lysis buffers typically contain non-ionic detergents (e.g., 1% Triton X-100)

• Protease inhibitor cocktails are essential to prevent degradation

• Samples should be denatured at 95°C for 5 minutes in reducing sample buffer

• Loading 20-50 μg of total protein per lane typically yields optimal results

For Immunohistochemistry:

• Formalin-fixed, paraffin-embedded (FFPE) tissues with antigen retrieval

• Recommended antigen retrieval: TE buffer pH 9.0

• Alternative: citrate buffer pH 6.0

• Antibody dilution: 1:200-1:500

For Immunofluorescence:

• PFA-fixed, Triton X-100 permeabilized samples

• Typical antibody concentration: 0.25-2 μg/mL

• 4% PFA fixation for 15 minutes at room temperature

• Permeabilization with 0.1-0.5% Triton X-100 for 10 minutes

These protocols have been validated across multiple studies and represent current best practices for EXOC8 antibody applications.

What are the appropriate positive controls for EXOC8 antibody validation?

Selection of appropriate positive controls is critical for meaningful EXOC8 antibody validation:

Cell Lines with Validated EXOC8 Expression:

• HEK-293 cells (confirmed by Western blot and IP)

• HeLa cells (confirmed by Western blot)

• U-2 OS cells (confirmed by immunofluorescence)

• HuH-7 cells (confirmed by Western blot)

• A431 cells (confirmed by immunofluorescence)

Tissue Types with High EXOC8 Expression:

• Human brain tissue (validated by Western blot)

• Mouse and rat testis tissue (validated by Western blot)

• Human placenta, cerebellum, and prostate (validated by immunohistochemistry)

For advanced applications, recombinant EXOC8 protein or cells transfected with EXOC8 expression constructs provide additional specificity controls. When using fusion protein constructs, researchers should be aware of potential tag influences on antibody binding, particularly for antibodies targeting regions near fusion points .

How can researchers troubleshoot non-specific binding with EXOC8 antibodies?

Non-specific binding commonly occurs with EXOC8 antibodies and requires systematic troubleshooting:

Common Sources of Non-Specificity:

• Antibody concentration – Excessive antibody concentrations amplify background signals. Begin with manufacturer-recommended dilutions and titrate as needed (typically 1:500-1:6000 for WB) .

• Blocking conditions – Insufficient blocking leads to high background. Optimize using:

  • 5% non-fat dry milk in TBST for Western blotting

  • 1-3% BSA for immunofluorescence applications

  • 5-10% normal serum (species different from primary antibody) for immunohistochemistry

• Wash stringency – Inadequate washing retains non-specific antibodies. Implement:

  • Increased wash duration (4-5 washes of 5-10 minutes each)

  • Higher detergent concentration in wash buffers (0.1-0.3% Tween-20)

• Cross-reactivity – EXOC8 antibodies may cross-react with structural homologs. Validate using:

  • Protein array screening against multiple targets

  • Antibodies targeting different EXOC8 epitopes to confirm results

For particularly challenging samples, consider protein A column purification followed by peptide affinity purification to enhance antibody specificity .

What is the current understanding of EXOC8's role in the exocyst complex assembly?

EXOC8 serves as a critical scaffolding component within the octameric exocyst complex:

The exocyst complex consists of eight subunits (EXOC1-EXOC8) that collectively regulate vesicular trafficking by mediating the tethering of secretory vesicles to the plasma membrane prior to SNARE-mediated fusion. EXOC8 specifically:

• Functions as a target of activated Ral subfamily GTPases, thereby regulating exocytosis

• Contributes to the structural scaffold that facilitates vesicle docking

• Plays an essential role in normal cortical development

Truncated EXOC8 protein, as observed in neurodevelopmental disorders, leads to improper assembly of the entire exocyst complex. This disruption results in the accumulation of neurotransmitters and other excretory vesicles within cells, particularly affecting neuronal function .

Recent studies have demonstrated that EXOC8 interacts with other exocyst components in a GTPase-dependent manner, with mutations in the EXOC8 gene causing mislocalization of the entire complex and subsequent cellular trafficking defects .

How can EXOC8 antibodies be effectively employed in colocalization studies?

EXOC8 colocalization studies require meticulous experimental design and antibody selection:

Recommended Protocol:

• Antibody Selection: Choose EXOC8 antibodies raised in different host species than antibodies against potential interacting partners to avoid secondary antibody cross-reactivity .

• Sample Preparation:

  • Fix cells with 4% PFA for 15 minutes

  • Permeabilize with 0.1-0.5% Triton X-100 for 10 minutes

  • Block with 1-3% BSA in PBS for 1 hour

• Antibody Application:

  • Apply EXOC8 antibody at appropriate dilution (1:50-1:500 for IF/ICC)

  • Include markers for cellular compartments of interest (e.g., plasma membrane, Golgi, vesicular markers)

  • Include DAPI for nuclear counterstaining

• Imaging Analysis:

  • Utilize confocal microscopy for optimal spatial resolution

  • Quantify colocalization using Pearson's or Mander's coefficients

  • Implement appropriate controls (single-stained samples, isotype controls)

Validated colocalization partners include plasma membrane markers, vesicular transport components, and other exocyst complex members. U-2 OS and A431 cells have been successfully used in EXOC8 colocalization studies .

What molecular weight variants of EXOC8 have been observed in different experimental conditions?

EXOC8 typically presents at 82 kDa, though several variants have been observed under different experimental conditions:

When unexpected bands appear, researchers should consider:

• Post-translational modifications (phosphorylation may cause migration shifts)

• Alternative splice variants

• Proteolytic degradation (especially in poorly preserved samples)

• Cross-reactivity with related proteins

For definitive identification, mass spectrometry analysis of immunoprecipitated bands can resolve ambiguities in molecular weight observations .

How should researchers design experiments to investigate EXOC8's role in neurodevelopmental disorders?

Investigating EXOC8's role in neurodevelopmental disorders requires integrated experimental approaches:

Genetic Analysis:

• Sequence EXOC8 in affected individuals and family members

• Focus on known pathogenic variants (e.g., c.1714G > T; p.Glu572Ter)

• Consider whole exome sequencing for novel variant discovery

Functional Validation:

• Generate cellular models with EXOC8 variants using CRISPR-Cas9

• Assess exocyst complex assembly using co-immunoprecipitation with other exocyst components

• Evaluate vesicular trafficking using live cell imaging with fluorescent cargo markers

Neurodevelopmental Assessment:

• Employ neural organoids derived from patient iPSCs

• Analyze cortical development patterns using immunohistochemistry

• Assess neurotransmitter vesicle accumulation using electron microscopy and EXOC8 immunogold labeling

Animal Models:

• Create conditional Exoc8 knockout mouse models

• Evaluate phenotypes including microcephaly, seizures, and brain atrophy

• Employ rescue experiments with wild-type EXOC8 expression

This multi-faceted approach enables correlation of genotype with cellular and organismal phenotypes, providing comprehensive insights into EXOC8's role in neurodevelopment .

What are the species reactivity profiles of commercially available EXOC8 antibodies?

Commercial EXOC8 antibodies exhibit distinct species reactivity profiles, requiring careful selection based on experimental models:

When selecting an antibody for cross-species applications:

• Verify epitope conservation across target species

• Consider antibodies raised against highly conserved regions for cross-reactivity

• Validate in each new species before conducting full experiments

• For evolutionary studies, select antibodies with the broadest validated species profile

What epitope considerations are important when selecting EXOC8 antibodies?

Epitope selection significantly impacts EXOC8 antibody performance and application suitability:

Key EXOC8 Regions and Corresponding Antibodies:

• N-Terminal Region: Several antibodies target this region, offering good accessibility in most applications

• Central Region (AA 360-389): Targeted by ABIN654161, suitable for Western blotting

• C-Terminal Region (AA 508-725): Multiple antibodies available, often accessible in native and denatured conformations

• Specific Fragment (AA 100-250): Targeted by Abcam ab254804, validated for IHC-P, WB, and ICC/IF

Advanced Epitope Considerations:

• Posttranslational modifications: May mask epitopes or alter antibody binding

• Protein interactions: Epitopes involved in protein-protein interactions may be inaccessible in co-complexes

• Domain structure: Functional domains may be preferentially targeted for specific applications

• Sequence conservation: Epitopes in highly conserved regions enable cross-species applications

For highest specificity, researchers can select antibodies targeting unique EXOC8 sequences with minimal homology to other exocyst components or related proteins .

How can researchers quantitatively assess EXOC8 expression levels across different experimental conditions?

Quantitative assessment of EXOC8 expression requires rigorous methodology and appropriate controls:

Western Blot Quantification:

• Load equal amounts of protein (20-50 μg) across all samples

• Include housekeeping controls (β-actin, GAPDH, tubulin)

• Utilize standard curves with recombinant EXOC8 for absolute quantification

• Employ densitometry software (ImageJ, Image Lab) for band intensity analysis

• Normalize EXOC8 signal to loading controls

qPCR for mRNA Expression:

• Design primers specific to EXOC8 (verify specificity against EXOSC8)

• Normalize to multiple reference genes (GAPDH, ACTB, 18S rRNA)

• Use the 2^-ΔΔCt method for relative quantification

Flow Cytometry for Cell-by-Cell Analysis:

• Permeabilize cells (0.1% saponin or 0.1% Triton X-100)

• Apply EXOC8 antibody at optimized concentration

• Include isotype controls to establish background fluorescence

• Analyze median fluorescence intensity across cell populations

Immunohistochemistry Quantification:

• Use automated image analysis software (QuPath, HALO)

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

• Calculate H-scores (0-300) by multiplying intensity by percentage

• Include positive and negative control tissues in each batch

When comparing across experimental conditions, maintain identical acquisition parameters and analyze all samples in parallel to minimize technical variation.

Disclaimer and Abbreviations

This document compiles research information about EXOC8 antibodies from diverse scientific sources. While comprehensive, researchers should consult primary literature and manufacturer specifications before designing experiments. The content represents current knowledge as of April 2025 but may be subject to revision as new research emerges.

Common Abbreviations:

• WB: Western Blot

• IHC-P: Immunohistochemistry-Paraffin

• IF/ICC: Immunofluorescence/Immunocytochemistry

• IP: Immunoprecipitation

• ELISA: Enzyme-Linked Immunosorbent Assay

• PFA: Paraformaldehyde

• BSA: Bovine Serum Albumin

• TBST: Tris-Buffered Saline with Tween-20