BTBD6 Antibody

What is BTBD6 and what cellular functions does it serve?

BTBD6 (BTB/POZ Domain Containing 6) is a protein that functions as part of the CUL3(KBTBD6/7) E3 ubiquitin ligase complex. It serves as a substrate adapter for the RAC1 guanine exchange factor (GEF) TIAM1, mediating its 'Lys-48' ubiquitination and proteasomal degradation . Through this mechanism, BTBD6 plays a critical role in regulating RAC1 signal transduction and downstream biological processes including cytoskeletal organization, cell migration, and cell proliferation . The protein contains a characteristic BTB (Broad-Complex, Tramtrack and Bric-a-brac) domain that facilitates protein-protein interactions, particularly within ubiquitin ligase complexes. BTBD6 is also known by several alternative names, including BDPL, BTB/POZ domain-containing protein 6, Lens BTB domain protein, and KBTB6/KBTBD6 .

What are the key applications for BTBD6 antibodies in research?

BTBD6 antibodies are versatile tools that can be employed across multiple research applications:

These applications allow researchers to investigate BTBD6 expression patterns, subcellular localization, protein-protein interactions, and functional roles in various biological contexts .

Which tissue and cell types commonly express BTBD6?

BTBD6 expression has been documented across multiple human tissues and cell types. Published research using validated antibodies has demonstrated BTBD6 expression in:

• Human brain tissue (used as positive control in Western blot)

• Human breast carcinoma tissue (detected via immunohistochemistry)

• HeLa cells (human cervical adenocarcinoma cell line)

• LOVO cells (human colon adenocarcinoma cell line)

Expression patterns may vary based on developmental stage, disease state, and cellular conditions, making BTBD6 antibodies valuable tools for comparative expression studies .

How do I select the appropriate BTBD6 antibody for my specific research application?

Selection of an appropriate BTBD6 antibody requires consideration of several key factors:

• Target epitope region: BTBD6 antibodies are available targeting various regions:

  • N-terminal region antibodies

  • C-terminal region antibodies

  • Middle region antibodies (e.g., GKAFNRCSHLTRHKKIHTAVKRYKCEECGKAFKRCSHLNEHKRVQRGEKS)

  • Internal region antibodies

  • Specific amino acid sequences (e.g., AA 87-136, AA 218-267)

• Species reactivity: Verify cross-reactivity with your experimental model:

  • Human-specific antibodies

  • Multi-species reactive antibodies (Human/Mouse/Rat)

  • Broader reactivity including zebrafish, cow, guinea pig, etc.

• Validation status: Prioritize antibodies with:

  • Published validation data in your application of interest

  • Recombinant expression validation

  • Verification in multiple applications

For novel research questions, consider using multiple antibodies targeting different epitopes to confirm findings and reduce epitope-specific artifacts .

What are the optimal storage and handling conditions for BTBD6 antibodies?

To maintain antibody integrity and performance, follow these research-validated guidelines:

• Storage temperature: Store at -20°C for long-term preservation

• Aliquoting: Divide into single-use aliquots to avoid repeated freeze-thaw cycles

• Short-term storage: 4°C is acceptable for temporary storage (1-2 weeks)

• Buffer conditions: Most BTBD6 antibodies are formulated in:

  • PBS with 0.09% sodium azide and 2% sucrose

  • PBS without Mg²⁺ and Ca²⁺, pH 7.4, with 150mM NaCl, 0.02% sodium azide and 50% glycerol

• Concentration: Typical working concentrations range from 0.05-1 mg/ml

Proper adherence to these guidelines will ensure consistent antibody performance across experiments and maximize shelf life .

What is the recommended protocol for Western blot detection of BTBD6?

For optimal Western blot detection of BTBD6, follow this research-validated protocol:

• Sample preparation:

  • Lyse cells in RIPA buffer with protease inhibitors

  • Quantify protein concentration (BCA or Bradford assay)

  • Load 20-40 μg total protein per lane

• Electrophoresis conditions:

  • Use 10-12% SDS-PAGE gels

  • BTBD6 migrates at approximately 45 kDa

• Transfer and blocking:

  • Transfer to PVDF membrane (recommended over nitrocellulose)

  • Block with 5% non-fat milk in TBST for 1 hour at room temperature

• Antibody incubation:

  • Primary: Anti-BTBD6 at 0.2-1.0 μg/ml in blocking buffer overnight at 4°C

  • Secondary: HRP-conjugated anti-rabbit IgG at 1:5000-1:10000 for 1 hour at room temperature

• Detection:

  • Develop using enhanced chemiluminescence substrate

  • Expected band size: 45 kDa

  • Validated positive control: Human brain lysate

This protocol has been validated for detection of endogenous levels of total BTBD6 protein .

How should I optimize immunohistochemistry protocols for BTBD6 detection in tissue sections?

For successful immunohistochemical detection of BTBD6 in tissue sections:

• Tissue preparation:

  • Use formalin-fixed, paraffin-embedded (FFPE) tissue sections (4-6 μm thickness)

  • Deparaffinize in xylene and rehydrate through graded alcohols

• Antigen retrieval (critical step):

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0)

  • Pressure cooker method (20 minutes) yields superior results

• Blocking and antibody incubation:

  • Block endogenous peroxidase with 3% H₂O₂

  • Protein block with 5% normal goat serum

  • Primary antibody: Anti-BTBD6 at 1:20-1:50 dilution

  • Incubate overnight at 4°C in a humidified chamber

• Detection system:

  • HRP-polymer detection system recommended over ABC method

  • DAB chromogen for visualization

  • Hematoxylin counterstain (light application)

• Controls:

  • Positive control: Human breast carcinoma tissue

  • Negative control: Primary antibody omission

  • Peptide competition for specificity verification

This protocol has been successfully used to detect BTBD6 in human breast carcinoma samples as demonstrated in published validation studies .

What are the critical considerations for immunofluorescence experiments using BTBD6 antibodies?

For high-quality immunofluorescence detection of BTBD6:

• Cell preparation:

  • Culture cells on glass coverslips or chamber slides

  • Fix with 4% paraformaldehyde (10 minutes, room temperature)

  • Permeabilize with 0.1% Triton X-100 (5 minutes)

• Blocking and antibody incubation:

  • Block with 5% normal serum (1 hour, room temperature)

  • Primary antibody: Anti-BTBD6 at 0.25-2 μg/ml or 1:100 dilution

  • Incubate overnight at 4°C

• Secondary antibody:

  • Alexa Fluor-conjugated secondary antibodies (1:500-1:1000)

  • Incubate 1 hour at room temperature in the dark

  • Counter-stain nuclei with DAPI

• Imaging considerations:

  • Use confocal microscopy for precise subcellular localization

  • Acquire Z-stacks for complete cellular distribution analysis

  • Include co-localization markers for organelle identification

• Controls and validation:

  • Validate in HeLa cells, which show characteristic BTBD6 staining patterns

  • Include secondary-only controls

  • Consider dual staining with CUL3 to confirm E3 ligase complex association

This methodology has been validated in published studies showing specific BTBD6 subcellular localization patterns .

How can BTBD6 antibodies be used to investigate the ubiquitin-proteasome pathway?

BTBD6 antibodies can be powerful tools for investigating ubiquitin-proteasome pathways through several advanced techniques:

• Co-immunoprecipitation (Co-IP) studies:

  • Use anti-BTBD6 antibodies to pull down BTBD6-containing complexes

  • Probe for CUL3, TIAM1, and other known interaction partners

  • Recommended buffer: 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 1 mM EDTA with protease inhibitors

  • Include MG132 treatment to stabilize ubiquitinated intermediates

• Sequential immunoprecipitation:

  • First IP: Anti-ubiquitin antibody

  • Second IP: Anti-BTBD6 antibody

  • This approach enriches for BTBD6-mediated ubiquitination events

• Proteasome inhibition experiments:

  • Treat cells with MG132 (10 μM, 4-6 hours)

  • Compare BTBD6 substrate levels (e.g., TIAM1) with/without inhibition

  • Use BTBD6 antibodies in Western blots to monitor complex formation

• In vitro ubiquitination assays:

  • Reconstitute CUL3(BTBD6) E3 ligase complex with purified components

  • Use anti-BTBD6 antibodies to confirm complex assembly

  • Detect substrate ubiquitination using anti-ubiquitin antibodies

These approaches leverage BTBD6 antibodies to investigate the protein's role in the ubiquitin-proteasome system, particularly its function as a substrate adapter in the CUL3(KBTBD6/7) E3 ubiquitin ligase complex .

What experimental approaches can be used to study BTBD6's role in RAC1 signaling pathways?

To investigate BTBD6's role in RAC1 signaling pathways, researchers can implement these approaches using BTBD6 antibodies:

• Active RAC1 pull-down assays:

  • Manipulate BTBD6 levels (knockdown/overexpression)

  • Use GST-PAK-CRIB domain to pull down active RAC1

  • Quantify active RAC1 levels by Western blot

  • Correlate with BTBD6 expression using anti-BTBD6 antibodies

• TIAM1 degradation kinetics:

  • Perform cycloheximide chase experiments

  • Track TIAM1 degradation with/without BTBD6 manipulation

  • Use anti-BTBD6 antibodies to confirm expression levels

  • Analyze by Western blot and quantitative image analysis

• GABARAP co-localization studies:

  • Perform dual immunofluorescence with anti-BTBD6 and anti-GABARAP antibodies

  • Quantify co-localization at membrane structures

  • Assess the impact of GABARAP manipulation on BTBD6 localization

• Migration and cytoskeletal phenotype analysis:

  • Conduct wound healing or transwell migration assays

  • Manipulate BTBD6 expression

  • Correlate migration phenotypes with RAC1 activity

  • Use phalloidin staining to visualize actin cytoskeleton

  • Confirm BTBD6 manipulation using validated antibodies

These methodologies leverage BTBD6 antibodies to elucidate the protein's role in regulating RAC1 signal transduction and downstream biological processes including cytoskeletal organization, cell migration, and cell proliferation .

How can cross-species conservation of BTBD6 function be studied using available antibodies?

To investigate the evolutionary conservation of BTBD6 function across species:

• Cross-species reactivity profiling:

  • Test validated BTBD6 antibodies against lysates from multiple species

  • Many antibodies show cross-reactivity with human, mouse, rat, cow, zebrafish, horse, guinea pig, and monkey samples

  • Confirm specific band detection at the predicted molecular weight for each species

• Comparative expression analysis:

  • Use antibodies with broad species reactivity

  • Perform parallel IHC or Western blots on tissues from different species

  • Compare expression patterns across developmental stages

  • Quantify relative expression levels

• Domain-specific antibody approach:

  • Select antibodies targeting highly conserved domains (e.g., BTB domain)

  • Perform epitope mapping to identify conserved binding regions

  • Use species-specific secondary antibodies for multiplexed detection

• Heterologous expression systems:

  • Express BTBD6 from different species in a common cellular background

  • Use antibodies to assess expression, localization, and complex formation

  • Perform functional rescue experiments across species

This multi-faceted approach using BTBD6 antibodies can reveal evolutionary conservation of protein function, expression patterns, and regulatory mechanisms across diverse species, providing insights into fundamental biological roles of this protein .

What are common issues encountered with BTBD6 antibodies and how can they be resolved?

Researchers may encounter several challenges when working with BTBD6 antibodies. Here are evidence-based solutions for common issues:

When troubleshooting, always include proper positive controls (e.g., human brain tissue for Western blot , breast carcinoma for IHC ) and validate findings with multiple BTBD6 antibodies targeting different epitopes .

How should contradictory results from different BTBD6 antibodies be interpreted?

When faced with contradictory results from different BTBD6 antibodies, follow this systematic approach for resolution:

• Epitope mapping comparison:

  • Compare the epitope regions of each antibody used

  • N-terminal, C-terminal, and internal region antibodies may reveal different aspects of protein regulation

  • Consider whether post-translational modifications might mask specific epitopes

• Validation hierarchy assessment:

  • Prioritize results from antibodies with:
    a) Recombinant expression validation
    b) Multiple application validation
    c) Published validation in your specific tissue/cell type

• Functional validation approaches:

  • Complement antibody studies with genetic approaches (siRNA, CRISPR)

  • Verify phenotypes match expected BTBD6 function in ubiquitin-proteasome regulation

  • Test for rescue of phenotypes with exogenous BTBD6 expression

• Confirmation via alternative methods:

  • Supplement antibody-based detection with mRNA analysis

  • Consider mass spectrometry validation for protein detection

  • Use epitope-tagged BTBD6 constructs as additional controls

• Biological context consideration:

  • Assess whether contradictions reflect true biological variation

  • Consider whether splicing variants might explain discrepancies

  • Evaluate cell-type specific expression patterns

This structured approach helps distinguish technical artifacts from biologically meaningful discoveries, facilitating accurate interpretation of complex BTBD6 data .

What quantitative approaches can accurately measure BTBD6 expression levels?

For rigorous quantification of BTBD6 expression levels, researchers can employ these methodologies:

• Western blot densitometry:

  • Use validated anti-BTBD6 antibodies at optimized concentrations (0.2-1.0 μg/ml)

  • Include titration curve with recombinant standards for absolute quantification

  • Normalize to multiple housekeeping proteins (β-actin, GAPDH, β-tubulin)

  • Use fluorescent secondary antibodies for wider linear detection range

  • Analyze with software like ImageJ or commercial alternatives

• Quantitative immunohistochemistry:

  • Employ standardized IHC protocols with BTBD6 antibodies at 1:20-1:50 dilution

  • Use automated staining platforms for consistency

  • Implement digital pathology analysis with algorithms for:
    a) H-score calculation (intensity × percentage positive cells)
    b) Optical density measurements
    c) Subcellular localization quantification

• ELISA-based quantification:

  • Develop sandwich ELISA using different epitope-targeting BTBD6 antibodies

  • Standard dilution: 1:10000 for detection antibody

  • Create standard curves using recombinant BTBD6 protein

  • Validate assay specificity with BTBD6-depleted samples

• Flow cytometry:

  • Optimize fixation and permeabilization for intracellular BTBD6 staining

  • Use fluorophore-conjugated anti-BTBD6 antibodies or two-step detection

  • Include isotype controls and fluorescence-minus-one controls

  • Quantify mean fluorescence intensity across cell populations