rabepk Antibody

What exactly is RABEPK and why is it important in cellular biology?

RABEPK (Rab9 Effector Protein with Kelch Motifs), also known as p40 or RAB9P40, functions as a Rab9 effector required for endosome to trans-Golgi network (TGN) transport. It is primarily localized in the cytoplasm and endosome membrane. Interaction with PIP5K3 and subsequent phosphorylation recruits it to the endosomal membrane . As a key component of endosomal trafficking pathways, RABEPK is critical for cellular homeostasis and membrane transport systems, making it an important target for researchers studying vesicular trafficking mechanisms.

How do rabbit-derived polyclonal RABEPK antibodies compare to mouse-derived monoclonal versions?

Rabbit-derived RABEPK antibodies typically offer several research advantages over their mouse counterparts:

• Enhanced specificity and affinity: The unique ontogeny of rabbit B cells produces antibody repertoires rich in in vivo pruned binders with high diversity, affinity, and specificity .

• Superior sensitivity: Multiple studies demonstrate that rabbit monoclonal antibodies show higher sensitivity compared to benchmark mouse monoclonal antibodies, particularly in immunohistochemistry applications .

• Epitope recognition: Rabbits recognize a broader range of epitopes as "foreign" compared to mice, resulting in more diverse binding capabilities .

• Light chain diversity: Rabbit antibodies feature highly diverse light chains with dominant roles in antigen binding, as evidenced by crystallography studies of rabbit antibody-antigen complexes .

What are the common applications for RABEPK antibodies in research settings?

RABEPK antibodies have been validated for multiple research applications:

What criteria should guide the selection of a RABEPK antibody for specific research applications?

When selecting a RABEPK antibody, researchers should consider:

• Target epitope region: Antibodies targeting different regions (N-terminal, central, or C-terminal) may yield different results. For example, ABIN2784781 targets the N-terminal region with the sequence "MKQLPVLEPG DKPRKATWYT LTVPGDSPCA RVGHSCSYLP PVGNAKRGKV" , while others target central or C-terminal regions.

• Species reactivity: Ensure compatibility with your experimental model. Some antibodies like ABIN2784781 offer broad cross-reactivity (Human, Mouse, Rat, Pig, Rabbit, Cow, Horse, Dog, Guinea Pig) , while others are species-specific.

• Application compatibility: Verify validation for your specific application. For instance, ABIN7247075 is validated for ELISA and IHC , while ABIN2856403 is validated for WB, ICC, IHC, IF, and IP .

• Clonality: Consider whether polyclonal (broader epitope recognition) or monoclonal (single epitope, higher consistency) better suits your experimental needs.

• Validation data: Review available validation data, including images of expected banding patterns, immunostaining patterns, and positive controls.

What validation experiments should researchers perform before using a new RABEPK antibody?

Comprehensive validation should include:

• Positive and negative control samples:

  • Use cell lines or tissues known to express or lack RABEPK

  • Include knockout/knockdown models when available

• Western blot analysis:

  • Verify the molecular weight (expected ~41 kDa for RABEPK)

  • Test multiple sample types and loading concentrations

• Peptide competition assay:

  • Pre-incubate antibody with immunizing peptide

  • Confirm signal reduction or elimination

• Cross-reactivity assessment:

  • Test across relevant species if conducting comparative studies

  • Verify predicted reactivity percentages (e.g., "Cow: 100%, Dog: 93%, Guinea Pig: 93%...")

• Method-specific validation:

  • For IHC: Test different fixation methods and antigen retrieval protocols

  • For IF: Confirm subcellular localization pattern matches known distribution

  • For IP: Verify enrichment of target protein in immunoprecipitated samples

How should sample preparation be optimized for RABEPK immunohistochemistry studies?

For optimal RABEPK detection in tissue sections:

• Fixation:

  • Formalin fixation (10% neutral buffered) for 24-48 hours is generally suitable

  • Overfixation may mask epitopes; consider shorter fixation times for sensitive epitopes

• Antigen retrieval:

  • Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Optimization may be required; test both buffers to determine optimal conditions

• Blocking:

  • Use 5-10% normal serum (species different from primary antibody host)

  • Consider specialized blocking reagents for high-background tissues

• Antibody dilution:

  • Start with manufacturer's recommendation (typically 1:50-1:300 for IHC)

  • Perform dilution series to optimize signal-to-noise ratio

• Incubation conditions:

  • Test both overnight incubation at 4°C and 1-2 hour incubation at room temperature

  • Ensure adequate humidity to prevent section drying

• Detection system:

  • Polymer-based detection systems often provide superior sensitivity

  • Consider tyramide signal amplification for low-abundance targets

What experimental parameters affect RABEPK antibody performance in Western blotting?

For optimal Western blot results with RABEPK antibodies:

• Sample preparation:

  • Use appropriate lysis buffers containing protease inhibitors

  • Consider phosphatase inhibitors if studying phosphorylation states

  • Optimal protein loading typically ranges from 20-30 μg total protein (as seen in ab137691 validation using 30 μg HepG2 lysate)

• Gel percentage and transfer conditions:

  • 10% SDS-PAGE is appropriate for resolving the 41 kDa RABEPK protein

  • Use semi-dry or wet transfer depending on protein size and hydrophobicity

• Blocking conditions:

  • Test both BSA and non-fat dry milk as blocking agents

  • Typically 5% blocking agent in TBST for 1 hour at room temperature

• Antibody dilution and incubation:

  • Follow manufacturer recommendations (typically 1:1000 for Western blot)

  • Incubate primary antibody overnight at 4°C for optimal results

• Washing and detection:

  • Thorough washing (3-5x with TBST) is critical for reducing background

  • ECL detection sensitivity should match expected protein abundance

How can researchers effectively study RABEPK interactions with other endosomal proteins?

To investigate RABEPK protein-protein interactions:

• Co-immunoprecipitation (Co-IP):

  • Use RABEPK antibodies validated for IP applications

  • Consider crosslinking approaches for transient interactions

  • Include appropriate negative controls (IgG, irrelevant antibody)

• Proximity ligation assay (PLA):

  • Enables visualization of protein interactions in situ

  • Requires antibodies from different host species against RABEPK and potential interacting proteins

• Immunofluorescence co-localization:

  • Use high-resolution confocal microscopy to assess spatial overlap

  • Consider super-resolution techniques for detailed subcellular localization

  • Quantify co-localization using appropriate statistical methods

• FRET/FLIM analysis:

  • For studying direct protein-protein interactions in live cells

  • Requires fluorescently tagged proteins

• Biochemical fractionation:

  • Isolate endosomal compartments using differential centrifugation

  • Analyze RABEPK distribution across fractions using Western blotting

What strategies can resolve weak or inconsistent signals in RABEPK immunostaining?

When encountering weak RABEPK immunostaining signals:

• Antibody concentration optimization:

  • Perform titration series to identify optimal concentration

  • Consider longer incubation times (overnight at 4°C)

• Antigen retrieval enhancement:

  • Extend heating time during antigen retrieval

  • Test alternative buffers (citrate pH 6.0 vs. EDTA pH 9.0)

  • Consider enzymatic retrieval for certain fixatives

• Signal amplification:

  • Implement tyramide signal amplification (TSA) system

  • Use biotin-streptavidin amplification methods

  • Consider polymer-based detection systems with enhanced sensitivity

• Tissue/cell preparation reassessment:

  • Evaluate fixation protocol impact on epitope preservation

  • Minimize time between tissue collection and fixation

  • Consider alternative fixatives for sensitive epitopes

• Antibody selection reconsideration:

  • Test antibodies targeting different epitopes of RABEPK

  • Compare N-terminal vs. C-terminal targeting antibodies

  • Consider switch between polyclonal and monoclonal antibodies

How can researchers distinguish between true RABEPK expression changes and technical artifacts?

To differentiate biological changes from technical variability:

• Implement multiple antibody validation:

  • Use at least two different antibodies targeting distinct RABEPK epitopes

  • Compare monoclonal and polyclonal antibody results

• Include comprehensive controls:

  • Positive controls (tissues/cells with known RABEPK expression)

  • Negative controls (knockout/knockdown samples)

  • Peptide competition controls to confirm specificity

• Employ orthogonal detection methods:

  • Validate immunostaining results with Western blotting

  • Confirm protein changes with mRNA expression analysis

  • Consider mass spectrometry for absolute quantification

• Standardize experimental conditions:

  • Process all comparative samples simultaneously

  • Use consistent reagent lots, incubation times, and temperatures

  • Include internal reference standards

• Quantitative analysis:

  • Use digital image analysis with appropriate normalization

  • Apply statistical methods to assess significance of observed differences

  • Implement blinded scoring by multiple observers for subjective assessments

What approaches enable the study of post-translational modifications of RABEPK?

For investigating RABEPK post-translational modifications:

• Phosphorylation studies:

  • Use phospho-specific antibodies when available

  • Combine with phosphatase inhibitors during sample preparation

  • Consider Phos-tag™ SDS-PAGE for mobility shift analysis

• Ubiquitination detection:

  • Immunoprecipitate RABEPK followed by ubiquitin Western blotting

  • Use deubiquitinase inhibitors during cell lysis

  • Consider tandem ubiquitin binding entity (TUBE) pulldown

• SUMOylation analysis:

  • Denaturing IP protocols to preserve SUMO modifications

  • Use SUMO-specific antibodies for Western blotting

  • Consider SUMO-protease inhibitors during sample preparation

• Mass spectrometry approaches:

  • Immunoprecipitate RABEPK for targeted MS analysis

  • Enrich for specific modifications using appropriate techniques

  • Apply parallel reaction monitoring for quantitative assessment

• Site-directed mutagenesis:

  • Generate RABEPK mutants at predicted modification sites

  • Assess functional consequences in cellular models

  • Compare wild-type and mutant localization and interactions

How should researchers quantitatively analyze RABEPK localization in subcellular compartments?

For rigorous analysis of RABEPK subcellular distribution:

• High-resolution imaging acquisition:

  • Use confocal microscopy with appropriate z-stack sampling

  • Consider super-resolution techniques for detailed localization

  • Maintain consistent acquisition parameters across samples

• Co-localization analysis with organelle markers:

  • Include established markers for endosomes, TGN, and other relevant compartments

  • Calculate Pearson's or Mander's coefficients for quantitative assessment

  • Use object-based methods for discrete structures

• Quantification approaches:

  • Measure fluorescence intensity in defined cellular regions

  • Determine percentage of RABEPK positive structures co-labeled with various markers

  • Assess changes in distribution following experimental manipulations

• Dynamic studies:

  • Implement live-cell imaging with fluorescently tagged RABEPK

  • Track movement between compartments over time

  • Measure rates of association/dissociation with specific organelles

• Statistical analysis:

  • Apply appropriate statistical tests based on data distribution

  • Include sufficient biological and technical replicates

  • Report effect sizes alongside p-values

How can researchers interpret contradictory results from different RABEPK antibodies?

When facing discrepancies between different RABEPK antibodies:

• Epitope mapping considerations:

  • Determine exact epitope regions recognized by each antibody

  • Consider protein conformation effects on epitope accessibility

  • Evaluate potential post-translational modifications within epitope regions

• Isoform specificity assessment:

  • Determine if antibodies recognize all or specific RABEPK isoforms

  • Review target sequences against known splice variants

  • Consider targeting different exons for variant-specific detection

• Technical validation:

  • Implement knockdown/knockout controls for each antibody

  • Perform peptide competition assays

  • Evaluate cross-reactivity with closely related proteins

• Context-dependent factors:

  • Assess if discrepancies are application-specific (e.g., WB vs. IHC)

  • Consider fixation or sample preparation impacts on epitope availability

  • Evaluate buffer conditions that might affect antibody performance

• Resolution strategies:

  • Generate consensus findings from multiple antibodies

  • Weight evidence based on validation stringency

  • Consider orthogonal methods that don't rely on antibodies

What are the implications of altered RABEPK expression patterns in disease models?

When interpreting RABEPK changes in pathological conditions:

• Functional pathway analysis:

  • Assess impact on endosome-to-TGN transport

  • Evaluate consequences for recycling of mannose-6-phosphate receptors

  • Investigate effects on lysosomal enzyme sorting

• Integration with other vesicular trafficking components:

  • Examine changes in Rab9 activation or localization

  • Assess associated adaptors and effectors in the same pathway

  • Consider compensatory mechanisms in the trafficking network

• Correlation with clinical parameters:

  • Associate RABEPK alterations with disease progression

  • Evaluate potential as biomarker for specific conditions

  • Correlate with response to therapies targeting vesicular transport

• Mechanistic studies:

  • Determine if changes are causative or consequential

  • Implement rescue experiments to establish direct relationships

  • Use genetic models to recapitulate observed changes

• Therapeutic implications:

  • Consider RABEPK pathway as potential intervention target

  • Assess specificity of targeting approaches

  • Evaluate potential for modulating RABEPK interactions