RABEPK Antibody

What is RABEPK and why is it significant in cellular research?

RABEPK (Rab9 effector protein with kelch motifs), also known as p40, is a 40-41 kDa protein that functions as an effector of Rab9 GTPase. It plays a critical role in endosome-to-trans-Golgi network (TGN) transport pathways, particularly in the trafficking of mannose 6-phosphate receptors. RABEPK predominantly interacts with the active form of Rab9 but does not interact with Rab7 or K-Ras, and fails to bind Rab9 when it's associated with GDI .

Approximately 30% of RABEPK is membrane-associated, while the remainder is found in the cytosol. This distribution pattern is particularly significant for researchers investigating vesicular trafficking mechanisms, as RABEPK serves as a potent transport factor in receptor trafficking systems .

What types of RABEPK antibodies are available for research applications?

Current research-grade RABEPK antibodies include:

Researchers should note that while most available antibodies demonstrate reactivity primarily with human samples, select antibodies show cross-reactivity with other species including mouse, rat, pig, rabbit, and other mammals .

How should I select the optimal RABEPK antibody for my specific research application?

Selection should be methodically approached based on:

• Application compatibility: Different antibodies perform optimally in specific applications:

  • For Western blot: Consider antibodies validated at 1:2000-1:6000 dilution (e.g., 15105-1-AP)

  • For IHC: Select antibodies with established performance at 1:50-1:800 (e.g., 10213-2-AP, HPA023920)

  • For IP applications: Choose antibodies validated specifically for immunoprecipitation

• Target region specificity: Select antibodies based on the domain you wish to study:

  • N-terminal targeting antibodies

  • C-terminal targeting antibodies

  • Internal region targeting antibodies

• Validation evidence: Prioritize antibodies with comprehensive validation data, particularly those validated using knockout controls, which represent the gold standard for specificity confirmation. This approach parallels methodologies used in recent Rab protein antibody validation studies .

What are the recommended protocols for RABEPK antibody validation in cellular systems?

A rigorous validation protocol for RABEPK antibodies should include:

• Knockout-based validation: Generate or obtain RABEPK knockout cell lines and compare antibody signals between wild-type and knockout cells in your application of interest .

• Mosaic validation approach for IF applications:

  • Plate wild-type and knockout cells together in the same well

  • Label different cell populations with distinct fluorescent dyes

  • Perform immunofluorescence staining and imaging in the same field of view

  • Quantify hundreds of cells for each condition to reduce bias

• Orthogonal validation:

  • Validate antibody specificity using multiple techniques (e.g., WB, IP, and IF)

  • Confirm results using multiple antibodies targeting different regions of RABEPK

  • Compare antibody signal with RNAseq data where available

What are the optimal dilution ranges for RABEPK antibody applications?

It is critical to note that optimal dilutions are sample-dependent and should be determined empirically for each experimental system .

How can I address specificity concerns when using RABEPK antibodies in my research?

To address specificity concerns:

• Perform critical controls:

  • Include positive control samples (e.g., HepG2, HeLa, K-562, Jurkat cells) known to express RABEPK

  • Include negative controls such as:

    • Primary antibody omission

    • Isotype controls

    • Ideally, RABEPK knockdown or knockout samples

• Cross-validate results with multiple antibodies:

  • Use antibodies targeting different epitopes of RABEPK

  • Compare polyclonal and monoclonal antibody results

  • Verify molecular weight (observed MW should be approximately 40 kDa)

• Confirm specificity using blocking peptides:

  • Pre-incubate antibody with excess immunizing peptide

  • Run parallel experiments with blocked and unblocked antibody

  • Signal elimination/reduction with peptide competition confirms specificity

This multi-faceted approach mirrors strategies used to validate other Rab protein antibodies in rigorous validation studies .

What factors might contribute to variability in RABEPK antibody performance across different experimental systems?

Several factors can impact antibody performance:

• Sample preparation variables:

  • Fixation methods (PFA vs. methanol) significantly impact epitope accessibility

  • Buffer composition can affect antibody-antigen interactions

  • Antigen retrieval methods (TE buffer pH 9.0 vs. citrate buffer pH 6.0) yield different results

• Technical factors:

  • Storage conditions (freeze-thaw cycles can degrade antibody functionality)

  • Incubation time and temperature variations

  • Detection system sensitivity differences (HRP vs. fluorescent conjugates)

• Biological variables:

  • RABEPK expression levels vary across cell types and tissues

  • Post-translational modifications may mask epitopes

  • Protein-protein interactions may sequester epitopes in certain cellular contexts

Researchers should systematically document these variables when troubleshooting inconsistent results .

How can RABEPK antibodies be optimally employed in co-localization studies with other Rab proteins?

For advanced co-localization studies:

• Antibody selection strategy:

  • Ensure host species differ between RABEPK and other Rab protein antibodies to avoid cross-reactivity

  • Verify spectral compatibility of secondary antibody fluorophores

  • Validate antibodies individually before attempting co-localization

• Optimized protocol:

  • Fix cells using 4% paraformaldehyde for 15 minutes at room temperature

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

  • Block with PBS containing 5% BSA, 5% goat serum, and 0.01% Triton X-100

  • Apply primary antibodies (validated RABEPK and Rab protein antibodies) in blocking buffer overnight at 4°C

  • Wash thoroughly (3 × 10 minutes) with IF buffer

  • Apply appropriate fluorophore-conjugated secondary antibodies (1.0 μg/mL) for 1 hour at room temperature

  • Include DAPI nuclear counterstain

• Analysis considerations:

  • Use confocal microscopy with appropriate controls for bleed-through

  • Employ quantitative co-localization coefficients (Pearson's, Manders')

  • Consider super-resolution techniques for detailed co-localization analysis

What are the challenges and solutions when using RABEPK antibodies for studying post-translational modifications?

Challenges:

• Epitope masking: Post-translational modifications can alter antibody recognition sites

• Specificity issues: Distinguishing modified from unmodified RABEPK

• Low abundance: Modified forms may represent a small fraction of total RABEPK

Solutions:

• Enrichment strategies:

  • Use phosphatase inhibitors for phosphorylation studies

  • Employ ubiquitin-binding domain pulldowns for ubiquitination studies

  • Consider RABEPK immunoprecipitation followed by modification-specific antibody detection

• Specialized antibodies:

  • Develop or obtain modification-specific antibodies (similar to phospho-Rab antibodies described in search result )

  • Validate these using appropriate controls (e.g., phosphatase treatment for phospho-specific antibodies)

• Complementary techniques:

  • Combine antibody-based detection with mass spectrometry

  • Use proximity ligation assays for enhanced sensitivity

  • Consider genetic approaches (e.g., mutation of modification sites)

This approach parallels successful strategies used in developing phospho-specific antibodies for related Rab proteins .

How can RABEPK antibodies be effectively used in studying disease-associated alterations in vesicular trafficking?

For disease-related studies:

• Tissue-specific considerations:

  • Use RABEPK antibodies validated specifically for human tissue samples

  • For cancer studies, consider antibodies validated in carcinoma tissues (e.g., lung squamous cell carcinoma)

  • Optimize antigen retrieval methods based on tissue type

• Quantitative analysis approaches:

  • Employ digital image analysis for IHC quantification

  • Consider multiplexed immunofluorescence to study RABEPK in context with other markers

  • Use flow cytometry for quantitative cell population analysis

• Experimental design for disease models:

  • Include appropriate disease and control samples

  • Consider temporal analyses to track RABEPK alterations during disease progression

  • Correlate RABEPK localization/expression with functional readouts of vesicular trafficking

This methodology aligns with recent studies suggesting connections between chromosome 9q33.3 (RABEPK locus) and pathophysiological stress conditions, particularly in lung cancer research contexts .