reep6 Antibody

What is REEP6 and why is it important in retinal research?

REEP6 (Receptor Expression Enhancing Protein 6) is a member of the REEP family proteins belonging to the YIP superfamily. It is specifically expressed in rod photoreceptors and plays a crucial role in the trafficking of proteins essential for phototransduction. REEP6 is required for correct function and survival of retinal photoreceptors, and mutations in the REEP6 gene are associated with autosomal recessive retinitis pigmentosa (RP) .

The significance of REEP6 in retinal research stems from its specialized role in:

• Maintaining endoplasmic reticulum and mitochondrial homeostasis in rod photoreceptors

• Facilitating the stability and/or trafficking of guanylate cyclases

• Potentially participating in clathrin-coated vesicle trafficking from the ER to the plasma membrane

• Regulating the expression of phototransduction proteins

What antibodies are commonly used for REEP6 detection in research settings?

For REEP6 detection, researchers frequently employ:

• Rabbit polyclonal antibodies that target recombinant fragments within human REEP6, typically amino acids 1-50

• Custom antibodies for specific experiments, such as those used in co-immunoprecipitation studies with anti-REEP6 antibodies capable of immunoprecipitating Clathrin from vesicle fractions

• Commercial antibodies like those from Proteintech (used at 1:2,000 dilution for western blotting)

These antibodies have been validated for multiple applications including:

• Immunohistochemistry on paraffin-embedded sections (IHC-P)

• Immunocytochemistry (ICC)

• Western blotting (WB)

• Immunoprecipitation (IP)

• Immuno-electron microscopy (Immuno-EM)

How can REEP6 antibodies be optimized for immunohistochemistry of retinal tissue?

Based on recent studies, optimization of REEP6 immunohistochemistry for retinal tissue involves:

Protocol Recommendations:

• Fixation: Use 4% paraformaldehyde (PFA) for tissue preservation

• Sectioning: Optimal thickness for retinal cryosections is typically 10-12 μm

• Antibody dilution: Anti-REEP6 antibodies are typically used at 1:100 to 1:200 dilution for immunostaining

• Permeabilization: PBS containing 0.2% Triton-X 100 is effective for accessing intracellular REEP6

• Secondary antibody: Alexa-conjugated secondary antibodies (e.g., Alexa594-conjugated goat-anti-rabbit at 1:300 dilution)

Subcellular Localization:
REEP6 is predominantly localized to the inner segment of photoreceptors, where it co-localizes with ER markers. This localization pattern serves as a positive control for antibody specificity, as demonstrated in multiple studies .

What are the recommended protocols for western blotting detection of REEP6?

For optimal western blotting detection of REEP6:

Sample Preparation:

• Retina lysate preparation: Brief sonication in 1× SDS loading buffer supplemented with protease inhibitor cocktail

• Protein resolution: 10% or 15% SDS-PAGE gels are suitable for REEP6 detection

• Transfer: Standard transfer to nitrocellulose membrane

Antibody Application:

• Primary antibody: Anti-REEP6 (1:2,000 dilution, Proteintech)

• Secondary antibody: HRP-conjugated goat anti-rabbit (1:5,000 dilution)

• Signal detection: ECL kit visualization

• Controls: GAPDH (1:10,000) as loading control

Representative Blot Analysis:
When properly executed, western blots should reveal a distinct band for REEP6 at approximately 20 kDa, completely absent in knockout models, confirming antibody specificity .

How can REEP6 antibodies be employed to investigate protein-protein interactions in photoreceptors?

REEP6 antibodies have proven valuable for elucidating protein-protein interactions through multiple approaches:

Co-immunoprecipitation Protocols:

• Vesicle fraction isolation: Differential centrifugation of retinal extracts to isolate membrane vesicles

• Immunoprecipitation: Using anti-REEP6 antibody to pull down interacting proteins

• Verification: Reverse co-IP with antibodies against suspected interacting partners

Demonstrated Interactions:
Studies have successfully used anti-REEP6 antibodies to demonstrate interactions with:

• Clathrin: Anti-REEP6 antibody immunoprecipitated Clathrin from vesicle fractions of bovine retina

• Syntaxin-3 (STX3): REEP6 interacts with STX3, suggesting a role in vesicle docking

Immuno-EM Applications:
Anti-REEP6 antibodies coupled with gold particles have successfully identified REEP6-positive vesicles with triskelion structures characteristic of Clathrin-coated vesicles, confirming REEP6's association with specific vesicle populations .

How do REEP6 antibody results differ between wild-type, heterozygous, and knockout mouse models?

Comparative analysis of REEP6 immunostaining across genotypes reveals important insights:

Wild-type (WT) Pattern:

• Strong immunoreactivity in rod photoreceptor inner segments

• Co-localization with ER markers

• Normal expression levels on western blot

Heterozygous (Reep6+/-) Pattern:

• Reduced but detectable REEP6 immunoreactivity

• Normal retinal morphology and function

• No apparent phenotypic defects despite reduced protein levels

Knockout (Reep6-/-) Pattern:

• Complete absence of REEP6 immunoreactivity by both immunohistochemistry and western blot

• Progressive retinal degeneration

• Decreased expression of transmembrane phototransduction proteins (rhodopsin, GC1, GC2)

Compound Heterozygous (Reep6L135P/-) Pattern:

• Mimics patient genotype with both a missense and a null allele

• Intermediate phenotype between heterozygous and homozygous knockout

• Suitable model for testing therapeutic interventions

How do researchers reconcile contradictory findings regarding REEP6's effect on phototransduction protein trafficking?

Recent studies have revealed inconsistencies in the reported effects of REEP6 deficiency on phototransduction proteins:

Contradictory Observations:

Methodological Resolution Approaches:

• Antibody validation: Ensure antibodies are detecting the correct epitopes with proper controls

• Knockout strategy comparison: Different CRISPR/Cas9 targeting strategies may result in differential effects

• Temporal analysis: Examine protein expression at multiple time points during degeneration

• Quantitative assessment: Use quantitative western blotting with proper normalization

• Secondary verification: Complement antibody-based detection with mRNA analysis (RNA-seq)

The 2025 study suggests that rather than complete absence of guanylate cyclases, REEP6 deficiency causes reduced expression of multiple transmembrane proteins, pointing to a broader effect on ER and Golgi function rather than specific trafficking defects .

What controls should be included when validating REEP6 antibody specificity?

For rigorous validation of REEP6 antibody specificity:

Essential Controls:

• Genetic knockout tissues: Complete absence of signal in Reep6-/- tissues provides definitive evidence of specificity

• Peptide competition: Pre-incubation of antibody with immunizing peptide should abolish specific signal

• Multiple antibodies: Using antibodies targeting different epitopes to confirm consistent patterns

• Cross-species validation: Testing antibody in tissues from multiple species where REEP6 is conserved

• Non-retinal tissues: Testing in tissues with known absence of REEP6 expression as negative controls

Validation Metrics:
Researchers should document and report:

• Signal-to-noise ratio in immunostaining

• Band specificity in western blots

• Reproducibility across technical and biological replicates

• Antibody lot-to-lot consistency

How can REEP6 antibodies be utilized to monitor gene therapy efficacy in retinal degeneration models?

REEP6 antibodies play a crucial role in evaluating gene therapy outcomes:

Therapeutic Monitoring Protocol:

• Dual immunostaining approach: Co-staining for both REEP6 and FLAG-tag (for tagged therapeutic REEP6) allows distinction between endogenous and exogenous protein

• Expression pattern analysis: Verify correct subcellular localization to the inner segment

• Treatment coverage assessment: Quantify percentage of retina showing REEP6 immunoreactivity after subretinal injection

• Functional correlation: Compare REEP6 expression patterns with ERG improvements and photoreceptor survival

Demonstrated Success:
In Reep6L135P/- mice treated with rAAV8-Reep6.1, researchers observed:

• Robust FLAG-tagged REEP6 expression colocalizing with REEP6 antibody staining

• Therapeutic expression extending to >70% of the retina

• Restoration of guanylyl cyclase 1 (GC1) expression in the outer segment

• Alleviation of ER stress response as evidenced by reduced caspase-12 activation

What advanced imaging techniques can enhance REEP6 antibody detection in subcellular compartments?

Innovative approaches to visualize REEP6 at the subcellular level include:

Super-resolution Microscopy:

• Stimulated emission depletion (STED) microscopy can resolve REEP6 localization within the crowded inner segment

• Single-molecule localization microscopy (PALM/STORM) enables precise mapping of REEP6 in relation to ER membranes

Correlative Light and Electron Microscopy (CLEM):

• Combining immunofluorescence with electron microscopy permits visualization of REEP6 in the context of ultrastructural details

• Gold-labeled REEP6 antibodies used for immuno-EM can reveal association with specific vesicle populations and membrane curvatures

Live-cell Imaging Applications:

• REEP6 antibody fragments (Fabs) can potentially be used for live imaging of REEP6 dynamics

• Expression of REEP6-GFP fusion proteins paired with antibody validation confirms proper localization and function

How might REEP6 antibodies contribute to understanding broader mechanisms of ER stress in retinal diseases?

REEP6 antibodies offer unique opportunities for investigating ER stress mechanisms:

Research Applications:

• Co-localization studies: Using REEP6 antibodies alongside ER stress markers (CHOP, caspase-12) to map stress propagation in degenerating photoreceptors

• Time-course analysis: Tracking REEP6 expression changes during unfolded protein response activation

• Therapeutic intervention assessment: Evaluating whether treatments alleviating ER stress restore normal REEP6 distribution

• Comparative disease models: Using REEP6 antibodies across different retinal degeneration models to identify common ER stress pathways

Translational Relevance:
Understanding REEP6's role in ER stress has broader implications for multiple retinal degenerative diseases that share ER stress mechanisms, potentially identifying common therapeutic targets .

What methodological advancements could improve quantitative analysis of REEP6 expression in disease models?

Future methodological refinements could include:

Emerging Quantitative Approaches:

• Multiplexed antibody imaging: Simultaneous detection of REEP6 with multiple phototransduction proteins

• Mass spectrometry immunoassays: Absolute quantification of REEP6 and interacting partners

• Single-cell analysis: Combining REEP6 antibody staining with single-cell transcriptomics

• Automated image analysis algorithms: Machine learning approaches for unbiased quantification of REEP6 localization patterns

• In situ proximity ligation assays: Quantifying REEP6 interactions with specific partners in intact tissue

Standardization Recommendations:
Researchers should adopt standardized reporting of:

• Antibody sources, catalog numbers, and dilutions

• Image acquisition parameters

• Quantification methods with statistical analysis

• Raw data availability to enable meta-analysis across studies