ZBTB46 Antibody, HRP conjugated

What is ZBTB46 and why is it significant in immunological research?

ZBTB46 (also known as BTBD4, ZNF340, BZEL) is a 589 amino acid protein containing one BTB/POZ domain. It functions as a transcriptional repressor for PRDM1 and is selectively expressed by conventional dendritic cells (cDCs) and their committed progenitors. Its significance lies in its specificity as a marker - ZBTB46 is not expressed by plasmacytoid DCs, monocytes, macrophages, or other lymphoid or myeloid lineages, making it valuable for distinguishing cDCs from other immune cell populations . While not required for cDC development, ZBTB46 represses the expression of alternative myeloid growth factor receptors, enforcing cDC lineage restriction .

What does HRP conjugation mean in the context of ZBTB46 antibodies?

HRP (Horseradish Peroxidase) conjugation refers to the covalent attachment of the enzyme to the ZBTB46 antibody. This conjugation enables direct detection in assays like Western blot and ELISA without the need for secondary antibodies. When exposed to an appropriate substrate, the HRP enzyme catalyzes a reaction resulting in a detectable signal (typically colorimetric, chemiluminescent, or fluorescent). For ZBTB46 antibodies, HRP conjugation provides a convenient one-step detection system, improving assay efficiency and potentially reducing background noise associated with secondary antibody binding .

What are the primary applications for ZBTB46 Antibody, HRP conjugated?

ZBTB46 Antibody, HRP conjugated is primarily used in the following applications:

• Western Blot (WB): Application dilutions typically range from 1:300-5000

• ELISA: Recommended dilutions range from 1:500-1000

• Immunohistochemistry (IHC): Used for detecting ZBTB46 in tissue sections, as demonstrated in human tonsil tissue

• Flow Cytometry: While not all HRP-conjugated versions are validated for flow cytometry, some ZBTB46 antibodies have been successfully used in this application

These applications allow researchers to detect and quantify ZBTB46 expression in cell lysates, tissue sections, and intact cells, providing valuable data on cDC identification and biology .

How should ZBTB46 Antibody, HRP conjugated be stored and handled to maintain optimal activity?

For optimal storage and handling of ZBTB46 Antibody, HRP conjugated:

• Storage temperature: Store at -20°C or -80°C upon receipt

• Aliquoting: Divide into multiple small aliquots to avoid repeated freeze-thaw cycles, which can degrade the antibody and reduce HRP activity

• Working solution: When preparing dilutions, use the recommended buffer systems (typically TBS with BSA or PBS with glycerol) at appropriate pH (typically pH 7.4)

• Shipping condition: Typically shipped at 4°C, but should be transferred to -20°C storage upon receipt

• Preservatives: Most formulations contain preservatives like 0.03% Proclin 300 to maintain stability

Proper storage and handling are critical for maintaining both antibody specificity and HRP enzymatic activity, ensuring consistent experimental results .

How can researchers validate the specificity of ZBTB46 antibody in their experimental system?

Validating ZBTB46 antibody specificity involves multiple complementary approaches:

• Positive and negative control samples:

  • Positive controls: Use cell lines or tissues known to express ZBTB46 (conventional dendritic cells, particularly cDCs)

  • Negative controls: Include samples from plasmacytoid DCs, monocytes, or macrophages which should not express ZBTB46

• Knockdown/knockout validation:

  • Perform siRNA knockdown or CRISPR knockout of ZBTB46 in positive control cells

  • Compare antibody reactivity between wild-type and knockdown/knockout samples

• Peptide competition assay:

  • Pre-incubate the antibody with the immunizing peptide (such as recombinant human ZBTB46 peptide fragments)

  • A specific antibody will show reduced or absent signal when pre-blocked with the immunizing peptide

• Cross-validation with multiple antibodies:

  • Use antibodies raised against different epitopes of ZBTB46

  • Consistent results across different antibodies increase confidence in specificity

• Molecular weight verification:

  • Verify that the detected protein is of the expected molecular weight (~64.1 kDa for ZBTB46)

  • Multiple bands may indicate splice variants, post-translational modifications, or non-specific binding

This multi-faceted validation strategy ensures that experimental findings truly reflect ZBTB46 biology and are not artifacts of non-specific antibody binding .

What are the potential cross-reactivity issues with ZBTB46 antibodies across species?

Cross-reactivity considerations for ZBTB46 antibodies include:

• Species-specific reactivity:

  • Most ZBTB46 antibodies are raised against human or mouse epitopes

  • Documented reactivity varies by product: some antibodies react with human only, others with mouse only, and some with both

  • Some antibodies are predicted to react with other species like rat, sheep, horse, and chicken based on sequence homology

• Epitope conservation analysis:

  • The target region of the antibody significantly affects cross-reactivity

  • Antibodies targeting highly conserved regions (e.g., the BTB/POZ domain) may show broader cross-reactivity

  • Those targeting variable regions may be more species-specific

• Experimental validation:

  • Even predicted reactivity requires experimental validation

  • Suppliers often indicate which cross-reactivity has been tested versus predicted based on homology

• Known limitations:

  • Some antibodies demonstrate species specificity that may limit translational research

  • For example, certain inhibitors show activity in human samples but not in murine models, which may parallel antibody behavior

Researchers should carefully select antibodies based on their target species and verify cross-reactivity claims experimentally before conducting cross-species comparative studies .

What are common troubleshooting strategies for weak or absent signals when using ZBTB46 Antibody, HRP conjugated in Western blot?

When encountering weak or absent signals with ZBTB46 Antibody, HRP conjugated in Western blot:

• Antibody concentration:

  • Try a range of dilutions (1:300-5000 is typically recommended)

  • For weak signals, use a higher concentration (1:300-1:500)

• Sample preparation:

  • Ensure complete protein denaturation and reduction

  • Verify protein loading (10-30 μg total protein per lane is typical)

  • Confirm sample integrity by running a housekeeping protein control

• Transfer conditions:

  • Optimize transfer time and voltage for high molecular weight proteins

  • Consider using low-methanol or methanol-free transfer buffers for better transfer of ZBTB46 (~64.1 kDa)

• Blocking conditions:

  • Try different blocking agents (BSA vs. non-fat milk)

  • Optimize blocking time (1-2 hours at room temperature or overnight at 4°C)

  • Note that the recommended storage buffer contains 1% BSA, suggesting BSA may be preferable

• Detection system:

  • Ensure the HRP substrate is fresh and properly prepared

  • Extend exposure time for weak signals

  • Try more sensitive detection substrates (enhanced chemiluminescence or SuperSignal)

• Protein expression level:

  • ZBTB46 may have variable expression levels across cell types

  • Consider concentrating samples from low-expressing cells

• Antibody storage:

  • Verify the antibody hasn't undergone multiple freeze-thaw cycles

  • Check for signs of precipitation or contamination

Systematically testing these variables while maintaining proper controls can help identify and resolve issues with weak or absent signals .

How can researchers optimize ZBTB46 Antibody, HRP conjugated for immunohistochemistry applications?

Optimizing ZBTB46 Antibody, HRP conjugated for immunohistochemistry:

• Tissue preparation:

  • Use freshly fixed tissues (10% neutral buffered formalin is standard)

  • Paraffin-embedded sections should be 4-6 μm thick

  • Optimize antigen retrieval methods (heat-induced epitope retrieval in citrate buffer pH 6.0 or EDTA buffer pH 9.0)

• Antibody dilution:

  • Start with the manufacturer's recommended dilution (typically 2 μg/ml)

  • Prepare a dilution series to determine optimal concentration

  • Balance specific signal versus background

• Incubation conditions:

  • Test both room temperature (1-2 hours) and 4°C (overnight) incubations

  • Humid chambers prevent section drying during incubation

• Signal amplification:

  • Direct HRP conjugation provides one-step detection

  • For weak signals, consider additional amplification systems (tyramide signal amplification)

• Counterstaining:

  • Use hematoxylin for nuclear counterstaining

  • Adjust counterstaining intensity to maintain ZBTB46 signal visibility

• Controls:

  • Include positive control tissue (human tonsil has been validated)

  • Include negative controls (omit primary antibody)

  • Include isotype controls at matching concentration

• Background reduction:

  • Block endogenous peroxidase activity (3% H₂O₂ in methanol, 10-15 minutes)

  • Use avidin/biotin blocking for tissues with endogenous biotin

  • Include protein blocking step (normal serum from same species as secondary antibody)

These optimization steps can help achieve specific staining of ZBTB46 in tissue sections while minimizing background and non-specific signals .

How can ZBTB46 antibodies be used to study dendritic cell development and differentiation?

ZBTB46 antibodies enable sophisticated analyses of dendritic cell development:

• Lineage tracing and identification:

  • ZBTB46 is expressed specifically in cDCs and their committed progenitors

  • Antibodies allow identification of this lineage in complex populations

  • Can distinguish true cDCs from other related myeloid cells (monocytes, macrophages)

• Differentiation studies:

  • Monitor ZBTB46 expression during DC development from hematopoietic precursors

  • Study extracellular vesicle (EV)-induced differentiation of monocytes into DCs

  • Investigate factors that induce or suppress cDC development

• Flow cytometry applications:

  • Multi-parameter flow cytometry using ZBTB46 antibodies enables:

    • Quantification of cDC populations in different tissues

    • Analysis of cDC subsets in inflammatory conditions

    • Sorting of pure cDC populations for functional studies

• Single-cell analysis:

  • Combine with other DC markers (HLA-DR, CD11c, BDCA1, CD1a) for comprehensive phenotyping

  • Track developmental trajectories from progenitors to mature cDCs

• Functional studies:

  • Correlate ZBTB46 expression with functional properties

  • Investigate how ZBTB46 represses alternative myeloid growth factor receptors

  • Study transcriptional networks in DC lineage commitment

• Disease models:

  • Examine DC development in disease settings

  • Track cDC populations in cancer, autoimmunity, and infection models

These applications leverage the specificity of ZBTB46 as a cDC marker to advance understanding of DC biology in health and disease .

What are the considerations when using ZBTB46 Antibody, HRP conjugated for multiplexed immunoassays?

Implementing ZBTB46 Antibody, HRP conjugated in multiplexed immunoassays requires addressing several technical considerations:

• Spectral overlap management:

  • HRP typically produces brown/DAB precipitate in chromogenic detection

  • For multiplexing, consider:

    • Sequential detection with different substrates (DAB, AEC, etc.)

    • Chromogenic multiplexing systems that produce different colors

    • Combining with fluorescent detection for spectral separation

• Primary antibody compatibility:

  • Ensure other primary antibodies are from different host species to avoid cross-reactivity

  • If using multiple rabbit antibodies, consider:

    • Directly conjugated antibodies with different reporters

    • Sequential detection with complete elution between rounds

    • Tyramide signal amplification with spectral unmixing

• Epitope accessibility:

  • Multiple rounds of antigen retrieval may be needed

  • Test different retrieval conditions compatible with all targets

  • Consider the order of antibody application (less abundant targets first)

• Signal-to-noise optimization:

  • HRP can create background through non-specific binding

  • Use appropriate blocking reagents for each detection system

  • Titrate antibody concentrations to minimize background

• Cross-reactivity prevention:

  • Perform single-stain controls to verify specificity

  • Include absorption controls if antibodies target related proteins

  • Block previously detected antibodies before subsequent rounds

• Data analysis:

  • Employ image analysis software for colocalization studies

  • Establish quantifiable metrics for each marker

  • Use appropriate statistical methods for multiplexed data

• Internal controls:

  • Include positive controls for each marker

  • Incorporate negative controls lacking one primary antibody at a time

These considerations help researchers successfully integrate ZBTB46 detection into multiplexed assays for comprehensive analysis of complex cell populations .

How do HRP-conjugated ZBTB46 antibodies compare to other conjugated formats (PE, FITC, AP) for different applications?

Comparative analysis of differently conjugated ZBTB46 antibodies:

Selection considerations:

• For Western blot and ELISA: HRP conjugates typically offer the best sensitivity and versatility

• For flow cytometry: PE conjugates provide superior brightness and are preferred

• For multiplex applications: Consider combining different conjugates with non-overlapping detection methods

• For archival samples: Chromogenic detection with HRP offers long-term stability compared to fluorescent conjugates

The choice between conjugates should be guided by the specific application, available detection instruments, and experimental design requirements .

What are the differences between monoclonal and polyclonal ZBTB46 antibodies, HRP conjugated, and when should each be used?

Comparing monoclonal and polyclonal ZBTB46 antibodies, HRP conjugated:

Selection guidelines:

• Choose polyclonal antibodies when:

  • Detecting low expression levels of ZBTB46

  • Working with partially denatured proteins

  • Maximum sensitivity is required

  • The exact epitope conformation is unknown

• Choose monoclonal antibodies when:

  • Consistent results across experiments are crucial

  • Specific domains of ZBTB46 need to be targeted

  • Minimal background is essential

  • Long-term studies require antibody consistency

• Application-specific considerations:

  • For Western blot: Both types work well, with monoclonals providing cleaner results

  • For IHC: Monoclonals may offer more specific staining patterns

  • For flow cytometry: Monoclonals are generally preferred for their specificity

The optimal choice depends on the research question, application, and whether specificity or sensitivity is the primary concern .

How can ZBTB46 antibodies be integrated into single-cell technologies for advanced immunophenotyping?

Integrating ZBTB46 antibodies into single-cell technologies:

• Single-cell RNA-seq validation:

  • Use ZBTB46 antibodies to sort cells for scRNA-seq

  • Validate transcriptomic clusters with protein-level ZBTB46 expression

  • Correlate ZBTB46 protein expression with transcriptional programs

• Mass cytometry (CyTOF) integration:

  • Metal-conjugated ZBTB46 antibodies can be incorporated into CyTOF panels

  • Enables simultaneous detection of 30+ markers including ZBTB46

  • Allows in-depth characterization of cDC subsets in complex tissues

• Imaging mass cytometry:

  • Combine ZBTB46 detection with spatial information

  • Map cDC distribution in relation to tissue microenvironments

  • Study cDC interactions with other immune and non-immune cells

• Spectral flow cytometry:

  • Include ZBTB46 in high-parameter flow panels (20+ colors)

  • Differentiate cDCs from similar populations using comprehensive marker panels

  • Apply advanced dimensionality reduction for visualization (t-SNE, UMAP)

• CITE-seq approaches:

  • Use oligonucleotide-tagged ZBTB46 antibodies for CITE-seq

  • Simultaneously measure ZBTB46 protein and transcriptome in single cells

  • Uncover relationships between ZBTB46 protein expression and gene expression programs

• Spatial transcriptomics correlation:

  • Validate spatial transcriptomics findings with ZBTB46 immunohistochemistry

  • Map ZBTB46+ cells to specific tissue niches

  • Correlate with gene expression patterns in the same regions

These cutting-edge applications enable comprehensive characterization of cDCs at unprecedented resolution, revealing heterogeneity and functional specialization within ZBTB46+ populations .

What are the emerging techniques for simultaneous detection of ZBTB46 protein expression and functional activity?

Emerging techniques for concurrent analysis of ZBTB46 expression and function:

• Transcription factor activity reporters:

  • Develop reporter systems responding to ZBTB46 transcriptional repression activity

  • Measure ZBTB46 binding to PRDM1 promoter regions while detecting protein expression

  • Correlate protein levels with functional repression activity

• Proximity ligation assays (PLA):

  • Detect ZBTB46 interaction with co-repressor complexes (SMRT/N-CoR-mSin3A HDAC complex)

  • Visualize protein-protein interactions in situ while quantifying expression levels

  • Map interaction networks in different DC subsets or activation states

• CRISPR-based functional screens with antibody detection:

  • Combine CRISPR screens targeting ZBTB46-associated factors

  • Use ZBTB46 antibodies to sort cells based on expression levels

  • Correlate genetic perturbations with protein expression and function

• Live-cell imaging with activity sensors:

  • Develop split fluorescent protein systems reporting on ZBTB46 activity

  • Monitor dynamic changes in localization and activity during DC development

  • Correlate with functional outputs like cytokine production

• ChIP-seq with simultaneous phenotyping:

  • Perform ChIP-seq using ZBTB46 antibodies

  • Correlate binding sites with expression levels in sorted subpopulations

  • Identify target genes and regulatory networks

• Multi-omics approaches:

  • Integrate ZBTB46 antibody-based sorting with proteomics and metabolomics

  • Correlate ZBTB46 expression with post-translational modifications

  • Link expression levels to metabolic states and functional capacity

• Functional genomics correlation:

  • Use ZBTB46 antibodies to isolate cells for ATAC-seq

  • Correlate chromatin accessibility with ZBTB46 expression

  • Map global epigenetic changes downstream of ZBTB46 activity

These advanced techniques enable researchers to move beyond simple detection toward understanding how ZBTB46 protein expression relates to its functional activity in regulating DC development and function .

How might ZBTB46 antibodies contribute to understanding the role of conventional dendritic cells in disease pathogenesis?

ZBTB46 antibodies can advance our understanding of cDC roles in disease through:

• Cancer immunology:

  • Precise identification of tumor-infiltrating cDCs using ZBTB46

  • Correlation of cDC infiltration patterns with patient outcomes

  • Study of cDC dysfunction in tumor microenvironments

  • Testing how treatments affect cDC populations and function

• Autoimmune disease:

  • Quantitative assessment of cDC populations in affected tissues

  • Characterization of cDC activation states in autoimmune conditions

  • Investigation of cDC-mediated antigen presentation to autoreactive T cells

  • Longitudinal monitoring of cDC populations during disease progression

• Infectious disease responses:

  • Tracking cDC responses to various pathogens

  • Studying pathogen-mediated manipulation of cDC function

  • Examining cDC migration patterns during infection

  • Identifying correlates of protective immunity in cDC populations

• Neurological disorders:

  • Investigating neuroimmune interactions involving cDCs

  • Studying cDC entry into the CNS during neuroinflammation

  • Examining cDC contributions to neurodegenerative diseases

• Therapeutic development:

  • Using ZBTB46 antibodies to monitor cDC-targeting therapies

  • Evaluating cDC responses to immunotherapies

  • Developing cDC-based vaccination strategies

  • Exploring extracellular vesicle (EV)-based approaches for DC manipulation

• Biomarker identification:

  • Assessing whether cDC phenotypes predict disease outcomes

  • Developing ZBTB46-based diagnostic or prognostic tools

  • Monitoring treatment efficacy through cDC population analysis

By specifically identifying cDCs using ZBTB46 antibodies, researchers can disentangle their unique contributions to disease pathogenesis from other myeloid populations, potentially leading to new therapeutic strategies targeting this specialized lineage .

What novel methodological approaches might enhance the utility of ZBTB46 antibodies in developmental immunology research?

Novel methodological approaches to enhance ZBTB46 antibody utility:

• Intravital imaging techniques:

  • Develop non-toxic fluorescent conjugates of ZBTB46 antibodies

  • Apply two-photon microscopy for deep tissue imaging of ZBTB46+ cells

  • Track cDC migration, interaction with T cells, and response to stimuli in vivo

• Organoid-based developmental systems:

  • Incorporate ZBTB46 antibody staining in lymphoid organoid cultures

  • Track cDC development in controlled microenvironments

  • Test factors that influence lineage commitment and differentiation

• CRISPR-engineered reporter systems:

  • Generate knock-in fluorescent proteins at the ZBTB46 locus

  • Validate reporter systems using ZBTB46 antibodies

  • Create dual reporter/knockout systems to study gene function

• Antibody engineering approaches:

  • Develop nanobody versions of ZBTB46 antibodies for improved tissue penetration

  • Create bispecific antibodies targeting ZBTB46 and functional markers

  • Engineer pH-sensitive fluorescent conjugates for endosomal tracking

• Advanced microfluidics integration:

  • Combine ZBTB46 antibody detection with microfluidic single-cell isolation

  • Develop lab-on-a-chip systems for cDC functional assessment

  • Create high-throughput screening platforms for factors affecting cDC development

• Genome-scale CRISPR screens:

  • Use ZBTB46 antibodies to sort cells after CRISPR perturbation

  • Identify genes regulating ZBTB46 expression and cDC development

  • Discover novel pathways controlling cDC lineage specification

• Extracellular vesicle research integration:

  • Study how EVs from mature DCs influence ZBTB46 expression in monocytes

  • Track EV-mediated transfer of molecules affecting ZBTB46 regulation

  • Develop therapeutic approaches based on EV manipulation of DC differentiation

These innovative approaches would significantly enhance our understanding of cDC development, function, and potential therapeutic applications by leveraging the specificity of ZBTB46 antibodies in increasingly sophisticated experimental systems .

What are the key specifications of commercially available ZBTB46 Antibody, HRP conjugated products?

Note: The table includes only HRP-conjugated ZBTB46 antibodies with detailed specifications available in the search results. Other conjugates (PE, FITC) and non-conjugated antibodies are available from additional suppliers .

What is the subcellular localization pattern of ZBTB46 and how does this affect experimental design?

ZBTB46 Subcellular Localization Data:

• Primary localization: Nuclear

• Functional context: Transcriptional repressor activity within the nucleus

• Domain structure: Contains BTB/POZ domain mediating protein-protein interactions and zinc finger domains for DNA binding

• Interaction partners: Associates with SMRT/N-CoR-mSin3A HDAC complex for gene silencing

Experimental Design Implications: