REPS1 Antibody

What is REPS1 and what cellular functions can be studied with REPS1 antibodies?

REPS1 is a ~87 kDa protein (653-744 amino acids) that coordinates the cellular actions of activated EGF receptors and Ral-GTPases . REPS1 antibodies allow researchers to investigate:

• Endocytosis and exocytosis pathways

• Receptor trafficking mechanisms

• Ras-Ral signaling pathway components

• Surface protein homeostasis regulation

REPS1 is widely expressed with particularly high levels in heart and testis tissue . Recent research has identified REPS1 as playing a crucial role in cargo exocytosis through a Reps1-Ralbp1-RalA module , making it an important target for cellular trafficking studies.

What validation methods should be employed to ensure REPS1 antibody specificity?

Proper validation of REPS1 antibodies is essential and should include:

• Knockout/knockdown verification: Testing on REPS1-KO cells is the gold standard. For example, researchers have validated antibodies using REPS1-KO HEK293E cells to confirm specificity of phospho-S709 REPS1 antibodies .

• Multiple detection methods: Cross-validate using different techniques:

  • Western blot (look for bands at ~80-110 kDa)

  • Immunoprecipitation followed by mass spectrometry

  • Immunocytochemistry with appropriate controls

• Peptide competition assays: Pre-incubation with immunizing peptides should abolish signal.

• Cross-reactivity testing: Confirm reactivity with expected species. Many REPS1 antibodies react with human, mouse, and rat samples, with approximately 90% gene identity between human and rodent REPS1 .

What are key differences between polyclonal and monoclonal REPS1 antibodies for research applications?

For investigating multiple REPS1 isoforms or ensuring detection of partially degraded protein, polyclonal antibodies may be preferable. For highly specific applications such as detecting post-translational modifications (e.g., phosphorylation at Ser709), monoclonal antibodies offer superior consistency .

How can researchers optimize protocols for studying REPS1 phosphorylation at Ser709?

REPS1 phosphorylation at Ser709 occurs within a RXRXXS/T motif, which is a consensus phosphorylation site for AGC kinases, particularly p90 ribosomal S6 kinase (RSK) . For optimal investigation:

• Use phospho-specific antibodies: Anti-phospho-S709 REPS1 antibodies are essential to distinguish phosphorylated from non-phosphorylated forms.

• Include proper controls:

  • Phosphatase treatment controls

  • S709A (serine-to-alanine) mutant as a negative control

  • Stimulate cells with RSK activators (e.g., PMA or EGF)

• Kinase inhibitor strategy:

  • Use selective RSK inhibitors (e.g., BI-D1870)

  • Include inhibitors for other AGC kinases (Akt, S6K1) as controls

  • Monitor phosphorylation changes by western blot

• Co-immunoprecipitation approach: To confirm direct interaction, perform co-IP experiments with REPS1 variants and RSK1. Previous research has shown that the C-terminal region of REPS1 has stronger binding to RSK1 than the N-terminal region .

• Sample preparation: Use phosphatase inhibitors (e.g., sodium orthovanadate, sodium fluoride) during cell lysis to preserve phosphorylation status.

What are the most effective applications of REPS1 antibodies in neurodegenerative disease research?

REPS1 has emerged as a potential biomarker in both Alzheimer's disease (AD) and vascular dementia (VD) . For neurodegenerative disease research:

• Tissue-specific analysis:

  • Use validated antibodies for human brain tissue sections

  • Compare REPS1 expression patterns between control and disease tissues

  • Analyze region-specific expression changes in brain

• Pathway investigation approaches:

  • Examine REPS1's relationship with Ras signaling pathway components

  • Focus on the negative correlation between REPS1 expression and Ras signaling, which is implicated in AD

  • Investigate REPS1's relationship with pyruvate metabolism and the citrate cycle, both linked to neuroinflammation and neurodegeneration

• Combined techniques:

  • Use immunohistochemistry for spatial distribution analysis

  • Couple with transcriptomic data for gene expression correlation

  • Perform co-localization studies with other AD/VD markers

• Biomarker validation:

  • Correlate REPS1 levels with clinical cognitive measures

  • Examine REPS1 in relation to established AD biomarkers (Aβ, tau)

  • Assess REPS1's potential as an early diagnostic marker

Research suggests REPS1 may aid in early diagnosis, monitoring of treatment response, and even efforts to prevent these debilitating disorders .

How can researchers effectively investigate the Reps1-Ralbp1-RalA pathway using REPS1 antibodies?

The Reps1-Ralbp1-RalA module plays a key role in cargo exocytosis and membrane trafficking . For studying this pathway:

• Protein complex analysis:

  • Use co-immunoprecipitation with REPS1 antibodies to pull down associated proteins

  • Confirm Ralbp1 interaction through western blot

  • Examine the effect of REPS1 knockout on Ralbp1 expression (typically strongly reduced)

• RalA activation assays:

  • Use GST pull-down assays with recombinant Ralbp1 to assess RalA binding

  • Quantify active RalA in wild-type versus REPS1-knockout cells (active RalA is typically reduced to near-background levels in REPS1-KO cells)

  • Examine total RalA levels by western blot for comparison

• Exocytosis measurement techniques:

  • Total internal reflection fluorescence microscopy (TIRFM) to monitor vesicle fusion events

  • Electron microscopy to detect vesicles near the plasma membrane

  • Surface biotinylation assays followed by mass spectrometry to analyze the surface proteome

• Functional recovery experiments:

  • Rescue experiments with wild-type versus mutant REPS1 expression

  • Specific domain deletion constructs to identify functional regions

  • Combined knockdown of pathway components to assess redundancy

Researchers should be aware that elimination of one subunit (e.g., REPS1) often leads to loss of other subunits (e.g., Ralbp1) in this complex .

What are common challenges in Western blot optimization with REPS1 antibodies and how can they be addressed?

Several issues can arise when using REPS1 antibodies in Western blot applications:

• Variable molecular weight detection: REPS1 has a calculated molecular weight of 71-81 kDa, but often appears at 80-110 kDa on gels . This discrepancy may be due to:

  • Post-translational modifications

  • Different isoforms (multiple splice variants)

  • High proline content affecting migration

  Solution: Include appropriate positive controls and use gradient gels (4-12%) to better resolve the protein.

• Weak signal strength:

  • Increase antibody concentration (typically 1:500-1:3000 for WB)

  • Extend primary antibody incubation to overnight at 4°C

  • Use enhanced chemiluminescence (ECL) detection system with longer exposure times

  • Consider signal amplification methods (e.g., biotin-streptavidin systems)

• High background:

  • Increase blocking time and concentration (5% BSA or milk)

  • Add 0.1-0.3% Tween-20 in wash buffers

  • Pre-adsorb antibody with non-specific proteins

  • Reduce secondary antibody concentration

• Sample preparation considerations:

  • Use freshly prepared samples when possible

  • Include protease inhibitors in lysis buffers

  • For phosphorylation studies, include phosphatase inhibitors

  • Optimal buffer: PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

How can researchers optimize immunofluorescence protocols for REPS1 localization studies?

For successful immunofluorescence experiments with REPS1 antibodies:

• Fixation optimization:

  • Test both paraformaldehyde (4%) and methanol fixation methods

  • For membrane-associated studies, avoid harsh permeabilization

  • Consider antigen retrieval methods if signal is weak

• Antibody dilution and incubation:

  • Start with manufacturer's recommended dilution and adjust as needed

  • Extend primary antibody incubation to overnight at 4°C

  • Use fluorophore-conjugated secondary antibodies with minimal spectral overlap with other channels

• Controls and validation:

  • Include REPS1 knockout or knockdown cells as negative controls

  • Compare staining pattern with published literature (primarily cytoplasmic with enrichment at membrane regions)

  • Consider co-staining with markers of subcellular compartments:

    • Early/recycling endosomal markers (e.g., EEA1, Rab11)

    • Plasma membrane markers

    • Clathrin-coated vesicle markers

• Signal enhancement for low abundance detection:

  • Use tyramide signal amplification systems

  • Consider confocal microscopy for better resolution of subcellular localization

  • For co-localization studies, use super-resolution microscopy techniques (STED, STORM)

• Image acquisition and analysis:

  • Capture z-stacks to ensure complete cellular visualization

  • Use deconvolution software to improve signal-to-noise ratio

  • Employ quantitative co-localization analysis software for interaction studies

How should researchers interpret contradictory findings when using different REPS1 antibodies?

When facing contradictory results with different REPS1 antibodies:

• Evaluate epitope differences:

  • Map the epitope locations of each antibody (N-terminal, C-terminal, or internal regions)

  • Consider that different antibodies may detect different isoforms or post-translationally modified variants

  • Check for epitope masking in protein complexes

• Assess validation strength:

  • Prioritize results from antibodies validated with knockout/knockdown controls

  • Review published validation data for each antibody

  • Consider performing your own validation experiments

• Cross-validation approaches:

  • Use alternative detection methods (e.g., mass spectrometry)

  • Employ tagged-REPS1 expression to compare with endogenous detection

  • Use siRNA knockdown to confirm specificity of each antibody

• Reconcile discrepancies through biological context:

  • Consider cell-type specific differences in REPS1 expression or modification

  • Evaluate experimental conditions that may affect epitope accessibility

  • Assess whether differences reflect physiologically relevant states of the protein

• Documentation and reporting:

  • Clearly document which antibody was used for which experiment

  • Report catalog numbers and lot numbers in publications

  • Describe detailed methods to allow reproduction by other researchers

How can REPS1 antibodies be used to explore its potential role as a biomarker in neurodegeneration?

To investigate REPS1 as a potential biomarker in neurodegeneration:

• Clinical sample analysis:

  • Compare REPS1 expression in post-mortem brain tissue from AD/VD patients vs. controls

  • Examine REPS1 levels in cerebrospinal fluid using sensitive ELISA methods

  • Correlate REPS1 levels with disease severity and progression

• Mechanistic investigation:

  • Study REPS1's relationship with microRNA hsa_miR_5701, which was predicted to regulate REPS1 expression

  • Investigate how REPS1 correlates negatively with infiltration by plasmacytoid dendritic cells in AD and VD

  • Examine connections between REPS1 and pyruvate metabolism, which REPS1 is predicted to activate

• Animal model studies:

  • Generate and characterize REPS1 knockout/knockin mouse models

  • Assess cognitive and behavioral phenotypes

  • Examine tissue-specific changes in REPS1 expression during disease progression

• Multi-omics integration:

  • Correlate REPS1 protein levels with transcriptomic data

  • Integrate with metabolomic studies focusing on pyruvate metabolism

  • Connect with proteomic analyses of Ras signaling components

Research indicates that REPS1 may specifically activate cellular redox homeostasis and pyruvate metabolism while inhibiting Ras signaling, all of which are implicated in neurodegenerative disease pathways .

What methodological approaches can researchers use to study REPS1's role in cargo exocytosis?

For investigating REPS1's function in cargo exocytosis:

• Live-cell imaging techniques:

  • Total Internal Reflection Fluorescence Microscopy (TIRFM) to visualize vesicle dynamics near the plasma membrane

  • Dual-color imaging to simultaneously track REPS1 and cargo proteins

  • High-speed imaging to capture rapid exocytic events

• Vesicle fusion assays:

  • pHluorin-based fusion reporters that fluoresce upon vesicle fusion with the plasma membrane

  • Transferrin recycling assays to measure receptor recycling rates

  • Biotinylation assays to quantify surface protein levels

• Electron microscopy approaches:

  • Transmission electron microscopy to detect vesicles near the plasma membrane

  • Immuno-gold labeling to visualize REPS1 localization at the ultrastructural level

  • Correlative light and electron microscopy for dynamic-structural correlation

• Functional perturbation strategies:

  • Compare wild-type versus REPS1-knockout cells using membrane protrusion assays

  • Use endocytosis inhibitors (e.g., ikarugamycin) to isolate exocytic events

  • Generate domain-specific mutants to identify critical regions for exocytosis

Research has shown that in REPS1-knockout cells, vesicles accumulate near the plasma membrane but fail to fuse properly, suggesting a specific role in the final steps of exocytosis .

How can researchers investigate the phosphorylation-dependent regulation of REPS1 function?

To study how phosphorylation affects REPS1 function:

• Phospho-mutant expression strategies:

  • Generate S709A (phospho-deficient) and S709D/E (phospho-mimetic) mutants

  • Compare cellular distribution and protein interactions of these mutants

  • Assess functional outcomes in trafficking assays

• Kinase manipulation approaches:

  • Use pharmacological inhibitors of RSK and other AGC kinases

  • Express constitutively active or dominant-negative kinase mutants

  • Perform in vitro kinase assays with purified components

• Phosphorylation dynamics:

  • Study temporal regulation using synchronized stimulation (e.g., growth factor treatment)

  • Use phospho-specific antibodies to track changes in REPS1 phosphorylation

  • Perform pulse-chase experiments to determine phosphorylation turnover rates

• Structural biology approaches:

  • Investigate how phosphorylation affects protein conformation

  • Examine crystal structures or use molecular modeling of phosphorylated versus non-phosphorylated forms

  • Assess changes in protein-protein interaction interfaces

• Functional correlations:

  • Determine how phosphorylation status affects cargo trafficking rates

  • Examine the relationship between REPS1 phosphorylation and RalA activation

  • Investigate phosphorylation-dependent changes in REPS1 interactome

Recent research has identified that RSK directly binds and phosphorylates REPS1, with the C-terminal region of REPS1 showing stronger binding to RSK1 than the N-terminal region .