ZBTB43 Antibody

What is ZBTB43 and why is it important in research?

ZBTB43 (Zinc Finger and BTB Domain Containing 43) is a protein that likely functions as a transcriptional regulator in the nucleus. It belongs to the zinc finger and BTB domain-containing protein family, which typically acts as transcription factors that regulate gene expression . The protein is also known by several synonyms including ZBT43, ZBTB22B, ZNF297B, and ZNFX .

Understanding ZBTB43 is important in research because BTB/zinc finger proteins often play critical roles in development, differentiation, and oncogenesis through their ability to modulate gene expression. The nuclear localization of ZBTB43 further supports its potential role in transcriptional control mechanisms . Research involving this protein may provide insights into gene regulation pathways and potential therapeutic targets.

What types of ZBTB43 antibodies are currently available for research?

Several types of ZBTB43 antibodies are currently available for research purposes:

• By host and clonality:

  • Rabbit polyclonal antibodies are the most common type available for ZBTB43 detection

  • These antibodies offer high sensitivity due to their recognition of multiple epitopes

• By target region:

  • Antibodies targeting specific amino acid regions such as AA 351-450

  • N-terminal specific antibodies

  • C-terminal specific antibodies

  • Full protein or larger segment antibodies (e.g., AA 165-467)

• By conjugation status:

  • Unconjugated primary antibodies

  • HRP-conjugated antibodies for direct detection without secondary antibodies

The diversity of available antibodies allows researchers to select the most appropriate tool based on their specific experimental needs and target tissues or species .

What are the common applications for ZBTB43 antibodies?

ZBTB43 antibodies can be utilized in multiple research applications:

• Western Blotting (WB): All the referenced antibodies support WB applications, making this the most common method for ZBTB43 protein detection and quantification . WB allows for determination of protein expression levels and apparent molecular weight.

• Immunohistochemistry (IHC):

  • IHC on paraffin-embedded sections (IHC-P)

  • IHC on frozen sections (IHC-fro)

  • Verified samples include human breast cancer and human liver cancer tissues

  • Recommended dilutions typically range from 1:50 to 1:300 for IHC applications

• ELISA: All referenced antibodies can be used in ELISA formats for quantitative detection of ZBTB43

• Immunocytochemistry/Immunofluorescence (ICC-IF): Some antibodies are validated for cellular localization studies

These applications enable researchers to investigate ZBTB43 expression, localization, and function in various experimental contexts, from cell culture to tissue samples .

How do I select the optimal epitope region when choosing a ZBTB43 antibody for my research?

Selecting the optimal epitope region requires careful consideration of several factors:

• Protein domain structure analysis:

  • The BTB domain (typically N-terminal) is often conserved across species and may provide broader cross-reactivity

  • Zinc finger domains (typically C-terminal) may offer more specificity to ZBTB43

  • Consider whether functional domains should be targeted or avoided based on your research question

• Post-translational modifications:

  • Determine if your region of interest contains potential phosphorylation, acetylation, or other modification sites

  • Modifications may block antibody binding, so choose epitopes that avoid known or predicted modification sites unless studying those specific modifications

• Species conservation:

  • For cross-species reactivity, select antibodies targeting highly conserved regions

  • Different antibodies show varying reactivity profiles, from human-specific to broadly reactive across species including mouse, rat, cow, dog, horse, and even Xenopus laevis

• Experimental context:

  • For detecting protein interactions, avoid epitopes that might be masked in protein complexes

  • For detection of protein fragments, ensure the epitope remains in your fragment of interest

  • N-terminal antibodies may be better for detecting truncated proteins that retain the N-terminus

Different commercial ZBTB43 antibodies target distinct regions (AA 151-200, AA 188-237, AA 351-450, N-term, C-term) , providing options to meet specific research requirements and experimental constraints.

What are the critical parameters for optimizing ZBTB43 antibody specificity in complex tissue samples?

Optimizing ZBTB43 antibody specificity in complex tissue samples requires attention to several critical parameters:

• Antibody validation controls:

  • Include positive controls (tissues/cells known to express ZBTB43)

  • Include negative controls (tissues/cells with minimal ZBTB43 expression)

  • Use blocking peptide controls to confirm specificity

  • Consider knockout/knockdown samples as gold-standard controls

• Sample preparation optimization:

  • For formalin-fixed samples, optimize antigen retrieval methods (heat-induced vs. enzyme-based)

  • For frozen sections, optimize fixation protocols to preserve antigenicity while maintaining tissue morphology

  • Test different blocking solutions to minimize background (BSA, normal serum, commercial blockers)

• Protocol adjustments:

  • Titrate antibody concentrations (recommended dilutions range from 1:50-1:300 for IHC)

  • Optimize incubation times and temperatures

  • Test different detection systems (amplified vs. non-amplified)

• Cross-reactivity mitigation:

  • Pre-absorb antibodies if cross-reactivity is observed

  • Use more stringent washing conditions

  • Apply tissue-specific blocking agents to reduce non-specific binding

• Signal enhancement strategies:

  • Consider tyramide signal amplification for low-abundance targets

  • Evaluate biotin-free detection systems to reduce endogenous biotin interference

Verified samples for ZBTB43 antibodies include human breast cancer and human liver cancer tissues, which can serve as reference points for optimization . Document all optimization steps systematically to establish reproducible protocols.

How can ZBTB43 antibodies be utilized to investigate protein-protein interactions and transcriptional complexes?

ZBTB43 antibodies can be powerful tools for investigating protein-protein interactions and transcriptional complexes through various advanced techniques:

• Co-immunoprecipitation (Co-IP):

  • Use unconjugated ZBTB43 antibodies to pull down ZBTB43 and associated proteins

  • Verify antibody compatibility with IP buffer conditions

  • Confirm the antibody epitope does not interfere with interaction domains

  • Cross-link antibodies to beads to avoid heavy chain contamination in subsequent analyses

• Chromatin Immunoprecipitation (ChIP):

  • Use ZBTB43 antibodies to identify DNA binding sites

  • Optimize formaldehyde crosslinking conditions

  • Verify antibody compatibility with ChIP buffer conditions

  • Consider dual ChIP approaches to identify co-occupancy with other factors

• Proximity Ligation Assay (PLA):

  • Combine ZBTB43 antibodies with antibodies against suspected interaction partners

  • Verify species compatibility of antibody pairs

  • Optimize fixation and permeabilization for nuclear proteins

• Immunofluorescence co-localization:

  • Use validated antibodies for ICC-IF applications

  • Employ high-resolution microscopy (confocal, super-resolution)

  • Quantify co-localization using appropriate statistical methods

• FRET/BRET analyses:

  • Use antibodies to validate interactions identified through resonance energy transfer approaches

  • Confirm epitope accessibility in fusion protein constructs

Given ZBTB43's nuclear localization and potential role in transcriptional regulation, these approaches can help elucidate its function within transcriptional complexes and its contribution to gene regulatory networks. When planning these experiments, consider using antibodies purified by protein A chromatography or antigen affinity purification for optimal performance .

What are the recommended protocols for using ZBTB43 antibodies in Western blotting applications?

For optimal Western blotting using ZBTB43 antibodies, follow these methodological recommendations:

• Sample preparation:

  • Use RIPA or NP-40 buffer supplemented with protease inhibitors

  • For nuclear proteins like ZBTB43, ensure efficient nuclear lysis

  • Include phosphatase inhibitors if phosphorylation states are important

  • Determine optimal protein loading (typically 20-50 μg of total protein)

• Gel electrophoresis and transfer:

  • Use 8-10% gels for optimal resolution of ZBTB43

  • Consider gradient gels for simultaneous detection of interaction partners

  • Optimize transfer conditions for large proteins (wet transfer, low SDS, longer transfer times)

  • Use PVDF membranes for better protein retention and signal

• Antibody incubation:

  • Block with 5% non-fat milk or BSA in TBST (optimize based on background)

  • For unconjugated antibodies, dilute according to manufacturer recommendations

  • For HRP-conjugated antibodies, adjust dilution to minimize background

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

  • Use compatible secondary antibodies for unconjugated primaries

• Detection and analysis:

  • For HRP-conjugated antibodies, proceed directly to detection

  • Use ECL substrate appropriate for expected expression level

  • For low expression, consider enhanced chemiluminescence substrates

  • Include positive controls and molecular weight markers

  • Perform densitometric analysis with appropriate normalization

• Validation steps:

  • Verify band size matches expected molecular weight

  • Consider peptide competition assays to confirm specificity

  • Include known positive samples when available

These methodological considerations should provide reliable detection of ZBTB43 protein while minimizing non-specific background and ensuring quantitative accuracy.

What protocols are recommended for immunohistochemistry applications with ZBTB43 antibodies?

For immunohistochemistry (IHC) applications with ZBTB43 antibodies, follow these protocol recommendations:

• Tissue preparation:

  • For FFPE tissues: Use standard fixation (10% neutral buffered formalin for 24-48 hours)

  • For frozen sections: Fix in cold acetone or 4% paraformaldehyde

  • Optimal section thickness: 4-6 μm for FFPE, 6-10 μm for frozen

• Antigen retrieval:

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

  • Optimize heating method (microwave, pressure cooker, or water bath)

  • Heat treatment time: 10-20 minutes after reaching boiling point

• Blocking and antibody incubation:

  • Block endogenous peroxidase with 3% H₂O₂ in methanol for 10 minutes

  • Block non-specific binding with 5-10% normal serum from secondary antibody host species

  • Apply primary antibody at 1:50-1:300 dilution as recommended

  • Incubate at 4°C overnight or at room temperature for 1-2 hours

  • For frozen sections, adjust antibody concentration as needed

• Detection system:

  • Use polymer-based or avidin-biotin detection systems

  • For direct detection, HRP-conjugated antibodies can be used

  • DAB substrate for brown staining, AEC for red (if needed for multiplexing)

  • Counterstain with hematoxylin for nuclear visualization

  • Mount with appropriate medium (aqueous for AEC, permanent for DAB)

• Controls and validation:

  • Include positive control tissues (human breast cancer and liver cancer tissues)

  • Include negative controls (primary antibody omission, isotype control)

  • Perform peptide competition if specificity concerns arise

  • Document staining intensity, pattern, and subcellular localization (nuclear for ZBTB43)

• Special considerations:

  • For multiplexing, use sequential staining or spectral unmixing approaches

  • For low expression, consider signal amplification systems (tyramide)

  • For double staining, ensure antibody compatibility (species, isotypes)

These protocol recommendations should provide optimal staining for ZBTB43 in tissue samples while minimizing background and non-specific signals.

How should researchers optimize storage and handling of ZBTB43 antibodies to maintain performance?

Proper storage and handling of ZBTB43 antibodies is critical for maintaining performance and extending shelf life:

• Storage conditions:

  • Store antibodies at -20°C for long-term storage as recommended by manufacturers

  • For antibodies containing 50% glycerol, storage at -20°C prevents freeze-thaw damage

  • Keep in small aliquots to minimize freeze-thaw cycles

  • For working stocks, store at 4°C for up to 2 weeks

  • Protect HRP-conjugated antibodies from light

• Freeze-thaw management:

  • Avoid repeated freeze-thaw cycles which can degrade antibody performance

  • Document number of freeze-thaw cycles for each aliquot

  • Thaw antibodies slowly on ice rather than at room temperature

  • Centrifuge briefly after thawing to collect contents

• Buffer considerations:

  • Maintain antibodies in manufacturer's buffer (typically phosphate buffered solution with stabilizers)

  • Avoid introducing contaminants (bacteria, fungi)

  • Consider adding sodium azide (0.02%) to unconjugated antibodies for extended 4°C storage

  • Never add preservatives to HRP-conjugated antibodies

• Shipping and temporary transport:

  • Transport with ice packs as recommended

  • Upon receipt, immediately transfer to appropriate storage temperature

  • Minimize time at room temperature

  • If shipping between labs, use dry ice for unconjugated antibodies

• Performance monitoring:

  • Include control experiments to monitor antibody performance over time

  • Keep a reference aliquot for comparison if performance issues arise

  • Document lot numbers and maintain records of performance

  • Consider regular validation with positive control samples

• Contamination prevention:

  • Use clean pipette tips for each access

  • Avoid touching the inside of the tube cap

  • Wear gloves when handling antibody containers

  • Centrifuge briefly before opening to collect condensation

Following these storage and handling recommendations should maintain ZBTB43 antibody performance for the full 12-month validity period indicated by manufacturers .

How can researchers troubleshoot non-specific binding when using ZBTB43 antibodies?

When encountering non-specific binding with ZBTB43 antibodies, implement the following troubleshooting approaches:

• Antibody-specific adjustments:

  • Optimize antibody concentration (titrate in wider ranges than recommended)

  • Try antibodies targeting different epitopes of ZBTB43

  • Consider antibodies purified by different methods (Protein A chromatography vs. antigen affinity)

  • For polyclonal antibodies showing high background, consider pre-absorption against tissues/cells lacking ZBTB43

• Protocol modifications:

  • Increase blocking stringency (longer time, higher concentration, alternative blockers)

  • Add detergents (0.1-0.3% Triton X-100 or Tween-20) to reduce hydrophobic interactions

  • Include protein additives like BSA, casein, or normal serum in antibody diluent

  • Increase wash duration and number of washes

  • Reduce primary antibody incubation temperature (4°C vs. room temperature)

• Sample preparation improvements:

  • Optimize fixation conditions (duration, fixative type)

  • For IHC, test alternative antigen retrieval methods

  • For WB, include reducing agents or chaotropes to minimize aggregation

  • Filter lysates to remove particulates that may bind antibodies non-specifically

• Background reduction strategies:

  • Block endogenous enzymes (peroxidase, phosphatase)

  • Use biotin-free detection systems if endogenous biotin is causing issues

  • For IHC, treat sections with avidin/biotin blocking kit if using ABC detection

  • For tissues with high Fc receptor content, include Fc receptor blocking step

• Controls for identifying non-specific binding:

  • Run isotype controls at the same concentration as primary antibody

  • Perform peptide competition assays

  • Include known negative tissues/cells

  • Test secondary antibody alone to identify secondary-mediated background

These troubleshooting approaches should help distinguish specific ZBTB43 signal from non-specific binding and optimize assay conditions for clear, interpretable results.

What are the considerations for interpreting ZBTB43 expression levels across different tissues and experimental conditions?

• Baseline expression establishment:

  • Document ZBTB43 expression in positive control tissues (breast cancer, liver cancer)

  • Create a reference panel of tissues with known expression levels

  • Consider both expression intensity and subcellular localization (nuclear)

  • Establish quantitative metrics for comparing expression across samples

• Technical normalization approaches:

  • For WB, normalize to appropriate loading controls (nuclear proteins for ZBTB43)

  • For qPCR validation, select stable reference genes

  • For IHC, use digital image analysis with standardized parameters

  • Account for batch effects when comparing samples processed separately

• Biological variability considerations:

  • Document donor-to-donor variability in human samples

  • Consider age, sex, and disease state influences on expression

  • For model organisms, account for strain differences

  • Evaluate cell-type-specific expression within heterogeneous tissues

• Experimental condition controls:

  • Include time-matched controls for temporal studies

  • For stress responses, include relevant control conditions

  • When using inhibitors or activators, include vehicle controls

  • For knockdown/overexpression studies, use appropriate vector controls

• Cross-method validation:

  • Confirm protein expression changes with mRNA levels

  • Use multiple antibodies targeting different epitopes

  • Apply orthogonal detection methods (WB, IHC, IF)

  • Consider absolute quantification methods for critical comparisons

• Functional context interpretation:

  • Correlate ZBTB43 expression with known targets or pathways

  • Consider protein modifications alongside expression levels

  • Evaluate changes in subcellular localization as well as total expression

  • Interpret expression changes in context of BTB-domain protein family members

These considerations provide a framework for robust interpretation of ZBTB43 expression data, enabling meaningful comparisons across experimental conditions and advancing understanding of its biological roles.

How can conflicting results from different ZBTB43 antibodies be reconciled in research studies?

When faced with conflicting results from different ZBTB43 antibodies, implement the following reconciliation strategies:

What are the emerging applications and future directions for ZBTB43 antibody research?

ZBTB43 antibody research is poised for several emerging applications and future directions:

• Single-cell analysis applications:

  • Adaptation of ZBTB43 antibodies for CyTOF/mass cytometry

  • Integration with single-cell genomics for multi-omic profiling

  • Development of high-sensitivity detection for low-abundance expression

  • Application in spatial transcriptomics for tissue-context understanding

• Therapeutic and diagnostic development:

  • Exploration of ZBTB43 as a biomarker in breast and liver cancers

  • Assessment of ZBTB43 expression in patient-derived xenografts

  • Correlation of expression with treatment response

  • Evaluation as a potential therapeutic target in transcriptional dysregulation

• Advanced imaging technologies:

  • Adaptation for super-resolution microscopy

  • Development of split-epitope approaches for proximity detection

  • Integration with live-cell imaging via nanobody development

  • Application in expansion microscopy for subcellular localization

• Cross-species comparative studies:

  • Leveraging broad species reactivity of some antibodies

  • Evolutionary conservation analysis of ZBTB43 function

  • Model organism validation of human findings

  • Development of species-specific antibodies for specialized applications

• Technological improvements:

  • Development of monoclonal antibodies for improved reproducibility

  • Creation of recombinant antibodies with defined epitopes

  • Engineered fragments for improved tissue penetration

  • Multiplexed detection systems for pathway analysis

• Functional characterization:

  • ChIP-seq applications to identify ZBTB43 binding sites

  • Proximity labeling to map protein interaction networks

  • Correlation with epigenetic marks in chromatin studies

  • Integration with CRISPR screens for functional genomics

The continued development and application of ZBTB43 antibodies will enable deeper understanding of this transcriptional regulator's role in normal development and disease processes, potentially opening new avenues for diagnostic and therapeutic interventions.

How should researchers evaluate and select ZBTB43 antibodies for reproducibility in long-term studies?

For ensuring reproducibility in long-term ZBTB43 studies, researchers should implement the following evaluation and selection strategies:

• Comprehensive initial validation:

  • Test multiple antibodies from different vendors

  • Evaluate different epitope targets (N-terminal, C-terminal, middle regions)

  • Perform specificity controls (peptide competition, KO/KD samples)

  • Document batch-to-batch consistency with reference samples

  • Generate validation data specific to your experimental system

• Long-term supply considerations:

  • Select antibodies with consistent manufacturing processes

  • Consider vendors with quality management systems (ISO certification)

  • Evaluate production scalability and supplier stability

  • Purchase larger lots when possible to minimize variation

  • Inquire about recombinant antibody options for ultimate consistency

• Documentation and standardization:

  • Create detailed antibody validation reports including lot numbers

  • Develop standard operating procedures (SOPs) for each application

  • Maintain electronic lab notebooks with all validation data

  • Establish minimal performance criteria for each application

  • Create reference sample banks for comparative testing

• Regular performance monitoring:

  • Implement scheduled revalidation experiments

  • Test each new antibody lot against reference samples

  • Monitor signal-to-noise ratios over time

  • Document any changes in staining patterns or intensity

  • Maintain positive control lysates/tissues with known ZBTB43 levels

• Redundancy planning:

  • Identify backup antibodies that perform similarly

  • Develop secondary detection methods as alternatives

  • Consider developing in-house antibodies for critical applications

  • Establish collaborations for antibody sharing and validation

• Publication and data sharing practices:

  • Report detailed antibody information in publications (catalog numbers, lots, dilutions)

  • Share validation data through repositories or supplementary materials

  • Participate in antibody validation initiatives

  • Contribute to community resources for antibody performance