RHOBTB3 Antibody

What is RHOBTB3 and what cellular functions does it regulate?

RHOBTB3 (Rho-related BTB domain-containing protein 3) is a 611 amino acid protein with a calculated molecular weight of 69 kDa that functions as a key regulatory protein in multiple cellular pathways. The protein directly interacts with the hydroxylase PHD2 to promote HIFα hydroxylation and also interacts with the von Hippel-Lindau (VHL) protein, facilitating ubiquitination of HIFα . RHOBTB3 serves as a scaffold to organize a multi-subunit complex (RHOBTB3/LIMD1-PHD2-VHL-HIFα) that promotes the hydroxylation, ubiquitination and degradation of HIFα, making it a critical regulator of cellular responses to hypoxia .

Beyond hypoxia regulation, RHOBTB3 functions as a Rab9-regulated ATPase required for endosome to Golgi transport. It is involved in transport vesicle docking at the Golgi complex, possibly by participating in the release of M6PRBP1/TIP47 from vesicles to permit their efficient docking and fusion at the Golgi . Importantly, RHOBTB3 specifically binds Rab9 but not other Rab proteins, and has low intrinsic ATPase activity due to autoinhibition, which is relieved by Rab9 .

What types of RHOBTB3 antibodies are available for research applications?

Several types of RHOBTB3 antibodies are available for research, differentiated by key characteristics:

The selection of the appropriate antibody depends on the specific research question, experimental technique, and target species. For cross-species studies, antibodies with broader reactivity profiles offer advantages, while region-specific antibodies may be critical for targeting particular functional domains of RHOBTB3 .

How do I determine optimal dilutions for different experimental applications?

Determining optimal dilutions for RHOBTB3 antibodies requires systematic titration across application-specific ranges:

For Western blot applications, the optimal dilution may vary depending on protein expression levels in different cell lines. For instance, RHOBTB3 has been successfully detected in K-562, U2OS, HEK-293, HeLa, and Jurkat cells as well as pig brain tissue using appropriate dilutions . For immunohistochemistry, antigen retrieval conditions significantly impact antibody performance, with TE buffer (pH 9.0) being recommended for optimal results, though citrate buffer (pH 6.0) may be used as an alternative .

What are the recommended storage conditions for maintaining RHOBTB3 antibody activity?

Proper storage is crucial for maintaining antibody functionality. RHOBTB3 antibodies should be stored at -20°C and remain stable for up to one year after shipment . Most RHOBTB3 antibodies are supplied in a storage buffer containing PBS with 0.02% sodium azide and 50% glycerol at pH 7.3, which helps maintain stability during freeze-thaw cycles .

For long-term storage and to minimize activity loss, consider these research-validated practices:

• Aliquot antibodies upon receipt to minimize freeze-thaw cycles (though some formulations specify that aliquoting is unnecessary)

• Avoid repeated freeze-thaw cycles as they can lead to protein denaturation and loss of binding activity

• Keep antibodies on ice when in use and return to -20°C promptly after use

• Monitor storage temperature stability, as temperature fluctuations can significantly reduce antibody shelf-life

• Check for signs of precipitation or contamination before use, as these indicate compromised antibody quality

How can I optimize Western blot protocols specifically for RHOBTB3 detection?

Optimizing Western blot protocols for RHOBTB3 detection requires attention to several critical variables:

Sample Preparation:

• Include protease inhibitors in lysis buffers to prevent RHOBTB3 degradation

• For subcellular localization studies, use fractionation protocols that preserve Golgi integrity, as RHOBTB3 is predominantly localized to the Golgi apparatus

• When studying hypoxia-related functions, prepare samples under controlled oxygen conditions to prevent artificial changes in RHOBTB3-HIFα interactions

Gel Electrophoresis and Transfer:

• Use 8-10% gels for optimal resolution of the 69 kDa RHOBTB3 protein

• Consider longer transfer times (90-120 minutes) for complete transfer of larger proteins

• For studying RHOBTB3 complexes, native PAGE may preserve protein-protein interactions better than SDS-PAGE

Detection Optimization:

• Use milk-based blocking solutions (3-5%) to minimize background

• For detecting endogenous RHOBTB3, longer primary antibody incubation (overnight at 4°C) often yields better results than shorter incubations

• When probing for interaction partners like PHD2 or VHL, consider sequential reprobing or parallel blots to avoid stripping artifacts

A validated protocol using mouse monoclonal RHOBTB3 antibody (67502-1-Ig) has demonstrated successful detection across multiple cell lines including K-562, U2OS, HEK-293, HeLa, and Jurkat cells, with optimal dilutions in the range of 1:1000-1:6000 .

What methodological approaches can resolve contradictory RHOBTB3 expression data?

Researchers frequently encounter contradictory expression data when studying RHOBTB3, which can be methodologically addressed through:

Antibody Validation Strategy:

• Use multiple antibodies targeting different epitopes (e.g., internal region vs. specific amino acid ranges like 251-500 or 1-99)

• Include positive controls (e.g., cells with verified RHOBTB3 expression) and negative controls (e.g., RHOBTB3 knockdown cells)

• Verify specificity using peptide competition assays to confirm epitope-specific binding

• Compare results across different detection methods (WB, IHC, IF) to triangulate true expression patterns

Addressing Experimental Variables:

• For hypoxia studies, standardize oxygen concentration and exposure times, as RHOBTB3 is functionally linked to hypoxia response pathways

• Account for cell-type specific expression patterns by comparing results across multiple cell lines

• Consider tissue-specific post-translational modifications that might affect antibody recognition

• Use qPCR to correlate protein expression with mRNA levels, particularly when antibody results are ambiguous

Advanced Verification Techniques:

• Mass spectrometry-based verification of RHOBTB3 presence in immunoprecipitated samples

• CRISPR/Cas9 knockout validation to confirm antibody specificity

• Recombinant expression systems for epitope mapping and antibody validation

How can I use RHOBTB3 antibodies to investigate its role in hypoxia signaling pathways?

RHOBTB3 plays a crucial role in hypoxia signaling through its interaction with PHD2 and VHL proteins to regulate HIFα degradation . To investigate these pathways:

Co-immunoprecipitation (Co-IP) Protocol:

• Use RHOBTB3 antibodies for immunoprecipitation from normoxic and hypoxic cell lysates

• Probe for interaction partners (PHD2, VHL, LIMD1, HIFα) in the precipitated complex

• Compare complex formation under different oxygen conditions, as hypoxia reduces RHOBTB3-centered complex formation

• Include appropriate controls (IgG control, input lysate)

Proximity Ligation Assay (PLA) Approach:

• Use RHOBTB3 antibody in combination with antibodies against PHD2, VHL, or HIFα

• Perform PLA in cells exposed to normoxia vs. hypoxia

• Quantify interaction signals to assess how oxygen levels affect protein-protein interactions

• Include single antibody controls to verify specificity

Functional Assays:

• Combine RHOBTB3 antibody staining with HIFα stability assays in cells under different oxygen conditions

• Monitor the Warburg effect parameters (e.g., lactate production, glucose consumption) in relation to RHOBTB3 expression levels

• Use immunofluorescence to track subcellular localization changes in response to hypoxia

• Correlate RHOBTB3 expression with tumor xenograft growth rates, as RHOBTB3 deficiency has been shown to accelerate xenograft growth

What considerations are important when using RHOBTB3 antibodies for immunohistochemistry?

Successful immunohistochemistry (IHC) with RHOBTB3 antibodies requires careful attention to several methodological factors:

Tissue Preparation and Antigen Retrieval:

• For formalin-fixed, paraffin-embedded (FFPE) tissues, TE buffer (pH 9.0) is recommended for optimal antigen retrieval, though citrate buffer (pH 6.0) can be used as an alternative

• Heat-induced epitope retrieval methods (pressure cooker or microwave) typically yield better results than enzymatic methods

• Optimization of retrieval time (10-30 minutes) may be necessary for different tissue types

Antibody Selection and Dilution:

• For mouse tissues (brain, heart), and rat adrenal gland tissue, monoclonal antibodies have shown reliable results at dilutions between 1:150-1:600

• For human tissues, both polyclonal and monoclonal antibodies can be effective, but optimization is tissue-dependent

• Consider using antibodies targeting the internal region of RHOBTB3 for broader species reactivity

Signal Detection and Specificity Controls:

• Implement positive controls (tissues with known RHOBTB3 expression) and negative controls (antibody diluent only)

• For tissues with low RHOBTB3 expression, amplification systems (e.g., tyramide signal amplification) may improve detection sensitivity

• When comparing normal and pathological tissues, standardize all staining parameters to ensure comparable results

• Consider dual staining with Golgi markers to confirm proper subcellular localization, as RHOBTB3 is primarily localized to the Golgi apparatus

How can I utilize RHOBTB3 antibodies to study its role in vesicular transport?

RHOBTB3 functions as a Rab9-regulated ATPase involved in endosome to Golgi transport . To study this role:

Colocalization Studies:

• Use RHOBTB3 antibodies in combination with markers for different cellular compartments (Rab9, TGN46, M6PRBP1/TIP47)

• Implement high-resolution microscopy techniques (confocal, STED, SIM) for precise localization

• Quantify colocalization coefficients in both static and dynamic trafficking assays

• Apply vesicle tracking methods to assess RHOBTB3's role in transport dynamics

Functional Transport Assays:

• Use RHOBTB3 antibodies to immunodeplete the protein from transport assays

• Monitor trafficking of mannose-6-phosphate receptors or other cargo between endosomes and Golgi

• Utilize RHOBTB3 function-blocking antibodies in permeabilized cell systems to assess transport inhibition

• Combine with Rab9 manipulation to explore the regulatory relationship between RHOBTB3 and Rab9

Biochemical Characterization:

• Use RHOBTB3 antibodies to isolate transport vesicles for proteomic analysis

• Implement ATPase activity assays with immunoprecipitated RHOBTB3 to study its intrinsic activity and regulation by Rab9

• Develop in vitro reconstitution assays to study RHOBTB3-dependent vesicle docking and fusion

• Use proximity labeling techniques with RHOBTB3 antibodies to identify novel interaction partners in the transport pathway

How can I troubleshoot common issues with RHOBTB3 antibody applications?

When encountering problems with RHOBTB3 antibody applications, systematic troubleshooting can identify and resolve issues:

For Western blot applications specifically, mouse monoclonal RHOBTB3 antibody (67502-1-Ig) has demonstrated consistent detection at the expected 69 kDa molecular weight across multiple cell lines, making it a reliable option for troubleshooting comparison .

What are the best practices for validating RHOBTB3 antibody specificity?

Comprehensive validation of RHOBTB3 antibody specificity is essential for generating reliable research data:

Primary Validation Methods:

• Genetic Controls: Test antibody reactivity in RHOBTB3 knockout/knockdown systems versus wild-type cells

• Recombinant Protein Controls: Use purified RHOBTB3 protein or overexpression systems as positive controls

• Peptide Competition: Pre-incubate antibody with immunizing peptide to block specific binding

• Multiple Antibodies: Compare results using antibodies targeting different epitopes (e.g., AA 335-384 versus internal region)

Application-Specific Validation:

• For Western blot: Verify correct molecular weight (69 kDa) and band pattern across multiple cell lines (K-562, U2OS, HEK-293, HeLa, Jurkat)

• For IHC/IF: Confirm expected subcellular localization (primarily Golgi apparatus) and compare staining patterns across different fixation methods

• For IP: Validate pull-down efficiency using Western blot and mass spectrometry to confirm target identity

Advanced Validation Approaches:

• Orthogonal method validation (comparing antibody-based detection with non-antibody methods)

• Isotype control experiments to distinguish specific from non-specific binding

• Cross-platform validation (comparing results across WB, IHC, IF, ELISA)