TOX2 Antibody, Biotin conjugated

What is TOX2 and what cellular functions does it regulate?

TOX2 (TOX High Mobility Group Box Family Member 2) is a transcription factor belonging to the TOX family that shares a highly conserved high mobility group DNA-binding domain with other TOX members. TOX2 plays critical roles in immune cell development, particularly in natural killer (NK) cells. Research has demonstrated that TOX2 is preferentially expressed in human NK cells among various leukocyte populations and is required for both in vitro and in vivo human NK cell differentiation from umbilical cord blood-derived CD34+ hematopoietic stem cells . The protein has a calculated molecular weight of approximately 52 kDa, though it is frequently observed at approximately 70 kDa in experimental conditions, possibly due to post-translational modifications . TOX2's transcriptional regulatory functions make it an important target for immunological research, particularly in studies investigating lymphocyte development and function.

What is the significance of biotin conjugation for TOX2 antibodies?

Biotin conjugation of TOX2 antibodies provides significant advantages in detection sensitivity and versatility across multiple applications. Biotinylation refers to the process of covalently linking biotin (vitamin H) to the antibody molecule, typically on amino acid residues or carbohydrate fractions . This conjugation creates a powerful tool for researchers because of the extremely high binding affinity between biotin and streptavidin/avidin proteins, characterized by a dissociation constant (KD) of approximately 1.3×10-15 M .

The biotin-streptavidin binding reaction offers several experimental advantages:

• Highly selective and rapid binding kinetics

• Amplified signal detection through multiple biotin binding sites on each streptavidin molecule

• Compatibility with various secondary detection systems

• Flexibility in experimental design through a wide range of streptavidin-conjugated detection molecules (fluorophores, enzymes, etc.)

These properties make biotin-conjugated TOX2 antibodies particularly valuable for detecting low-abundance transcription factors in complex biological samples with enhanced sensitivity .

What applications are optimal for using TOX2 Antibody, Biotin conjugated?

TOX2 Antibody, Biotin conjugated can be employed across multiple experimental platforms, with optimization considerations for each application:

For all applications, it is recommended to titrate the antibody concentration for each specific experimental system to achieve optimal signal-to-noise ratios. The biotin conjugation allows detection using streptavidin-coupled reporter molecules, which provides flexibility in experimental design and potential signal amplification .

How should TOX2 Antibody, Biotin conjugated be stored and handled to maintain activity?

Proper storage and handling of TOX2 Antibody, Biotin conjugated is critical to maintain its activity and specificity:

• Storage temperature: Store at -20°C or -80°C as indicated by the manufacturer

• Buffer composition: Typically provided in PBS with 0.02% sodium azide and 50% glycerol at pH 7.4

• Aliquoting: For -20°C storage, aliquoting may be unnecessary for some formulations, though it is generally recommended to minimize freeze-thaw cycles

• Thawing procedure: Thaw on ice and briefly centrifuge before opening to collect all material

• Stability: Most formulations remain stable for at least one year when stored properly

• Avoid repeated freeze-thaw cycles: These can lead to denaturation and loss of binding activity

• Working dilution preparation: Prepare fresh working dilutions on the day of the experiment

Following these guidelines will help preserve antibody performance and extend the usable life of the reagent in research applications .

What validation methods should be employed to confirm specificity of TOX2 Antibody, Biotin conjugated?

Comprehensive validation of TOX2 Antibody, Biotin conjugated specificity requires multiple complementary approaches:

• Positive and negative control samples:

  • Positive controls: A549 cells, HepG2 cells, HeLa cells, HEK-293 cells, MCF-7 cells, mouse/rat liver tissue

  • Negative controls: Cell lines with confirmed low/no TOX2 expression or TOX2 knockout models

• Blocking peptide competition assay:

  • Pre-incubate antibody with excess immunizing peptide (e.g., recombinant TOX2 protein fragments)

  • Compare signal between blocked and unblocked antibody conditions

• siRNA or CRISPR knockdown validation:

  • Generate TOX2 knockdown/knockout cells

  • Confirm reduction/absence of signal in Western blot or flow cytometry

• Cross-reactivity assessment:

  • Test antibody against recombinant proteins of related TOX family members

  • Verify absence of non-specific binding in tissues from different species

• Molecular weight verification:

  • Confirm detection at the expected molecular weight (~70 kDa observed for TOX2)

  • Verify any additional bands with appropriate controls

• Correlation with mRNA expression:

  • Compare protein detection levels with RT-PCR or RNA-seq data

How can biotin-conjugated TOX2 antibody be integrated into multiplex immunoassays?

Integrating biotin-conjugated TOX2 antibody into multiplex immunoassays requires careful consideration of several technical factors:

• Streptavidin reporter selection:

  • Choose a streptavidin conjugate with a detection wavelength that minimizes spectral overlap with other fluorophores

  • Consider quantum dots or spectrally distinct fluorophores for improved separation

• Multiplexing strategies:

  • Sequential detection: Apply biotin-conjugated TOX2 antibody after other directly-labeled antibodies

  • Use streptavidin conjugates with distinct reporters (e.g., different fluorophores or enzymes)

  • Consider tyramide signal amplification (TSA) for enhanced sensitivity

• Blocking endogenous biotin:

  • Implement avidin/streptavidin blocking steps before applying biotin-conjugated antibodies

  • Use commercial biotin blocking kits specifically designed for multiplexed assays

• Cross-reactivity prevention:

  • Pre-adsorb antibodies against tissues/cells of interest

  • Use isotype-specific secondary detection systems

  • Include appropriate blocking agents (e.g., serum, BSA) at optimized concentrations

• Signal separation and compensation:

  • For flow cytometry: perform proper compensation controls

  • For imaging: use spectral unmixing algorithms and single-stained controls

The biotin-streptavidin system provides excellent signal amplification in multiplex assays due to its high binding affinity (KD of 1.3×10-15 M) and the tetrameric nature of streptavidin, which can bind multiple biotin molecules, enhancing detection sensitivity of low-abundance nuclear factors like TOX2 .

What strategies can address interference from endogenous biotin when using TOX2 Antibody, Biotin conjugated?

Endogenous biotin can significantly interfere with detection systems using biotin-conjugated antibodies, particularly in tissues with high biotin content (e.g., liver, kidney, brain). Several strategies can mitigate this issue:

• Avidin/streptavidin blocking:

  • Pre-block tissues with unconjugated avidin/streptavidin

  • Follow with biotin solution to occupy remaining binding sites

  • Complete with a final avidin/streptavidin step to block remaining endogenous biotin

• Alternative fixation and antigen retrieval:

  • Use fixatives that reduce biotin accessibility (e.g., modified PFA protocols)

  • Optimize antigen retrieval methods that minimize endogenous biotin exposure

• Biotin-free detection systems:

  • Consider alternative approaches like directly labeled secondary antibodies if biotin interference cannot be adequately controlled

  • Use polymer-based detection systems as an alternative

• Special considerations for brain tissue:

  • For brain-specific applications, endogenous biotin may be particularly problematic as biotin is actively transported across the blood-brain barrier via specific transport mechanisms

  • The OX26 monoclonal antibody to the transferrin receptor undergoes transcytosis through the brain capillary endothelial wall, which can complicate biotin-based detection systems in brain tissue

• Tissue-specific blocking optimization:

  • Increase concentration and duration of blocking steps for tissues known to have high endogenous biotin

  • Include additional quenching steps before primary antibody application

These approaches should be empirically tested for each specific tissue type and experimental condition when using biotin-conjugated TOX2 antibodies .

How can researchers optimize TOX2 Antibody, Biotin conjugated for studying TOX2's role in immune cell development?

Optimizing TOX2 Antibody, Biotin conjugated for immune cell development studies requires specific methodological considerations:

• Cell isolation and preparation protocols:

  • Use gentle cell isolation methods to preserve native TOX2 expression levels

  • For NK cell studies, consider magnetic isolation or flow cytometry sorting with minimal manipulation

  • Optimize fixation and permeabilization protocols for intracellular transcription factor detection

• Developmental time-course analyses:

  • Design experiments to capture TOX2 expression at critical developmental stages

  • Correlate TOX2 levels with functional markers of cell differentiation

  • Consider paired analysis of TOX2 with other transcription factors involved in lymphocyte development

• Quantitative assessment methods:

  • Flow cytometry: Use 0.25 μg antibody per 10^6 cells for intracellular staining

  • Implement fluorescence minus one (FMO) controls for accurate gating

  • Consider phospho-flow techniques if studying signaling pathways upstream or downstream of TOX2

• Co-localization studies:

  • Combine TOX2 detection with lineage-specific markers

  • Use confocal microscopy with appropriate nuclear counterstains

  • Implement quantitative co-localization analysis (e.g., Pearson's correlation coefficient)

• Functional correlation:

  • Design assays to correlate TOX2 expression with functional outputs (e.g., cytokine production, cytotoxicity)

  • Consider paired TOX2 detection with functional readouts at the single-cell level

Previous research demonstrated that TOX2 is preferentially expressed in human NK cells and is required for NK cell differentiation from hematopoietic stem cells, making TOX2 antibodies valuable tools for studying NK cell development pathways .

What are effective troubleshooting strategies for non-specific binding when using TOX2 Antibody, Biotin conjugated?

Non-specific binding is a common challenge when working with biotin-conjugated antibodies. The following troubleshooting strategies can help resolve these issues:

• Optimization of blocking conditions:

  • Test different blocking agents (BSA, normal serum, commercial blockers)

  • Increase blocking time and concentration

  • Consider adding 0.1-0.3% Triton X-100 or Tween-20 to reduce hydrophobic interactions

• Titration and dilution optimization:

  • Perform careful antibody titration experiments (recommended range: 1:5000-1:50000 for Western blot)

  • Reduce primary antibody concentration if background is high

  • Optimize streptavidin-conjugate concentration independently

• Buffer modifications:

  • Increase salt concentration in wash buffers (150-500 mM NaCl)

  • Add 0.01-0.05% SDS to reduce hydrophobic interactions

  • Consider adding 5-10% normal serum from the host species of secondary reagent

• Pre-adsorption procedures:

  • Pre-adsorb the antibody against tissues or cells lacking TOX2

  • Implement commercial antibody pre-adsorption kits if available

• Streptavidin detection system adjustments:

  • Reduce streptavidin-conjugate concentration

  • Implement additional washing steps before and after streptavidin incubation

  • Consider using streptavidin conjugates with less background (e.g., streptavidin-Alexa Fluor conjugates)

• Sample-specific considerations:

  • For tissue sections, increase section thickness to improve signal-to-noise ratio

  • For cells in suspension, optimize fixation and permeabilization protocols

  • For Western blotting, increase washing duration and number of washes

When troubleshooting, modify one parameter at a time and maintain appropriate controls to accurately assess improvements in signal-to-noise ratio .

How does the performance of biotin-conjugated TOX2 antibody compare with direct fluorophore conjugates in advanced imaging applications?

In advanced imaging applications, biotin-conjugated TOX2 antibody and direct fluorophore conjugates each offer distinct advantages and limitations:

For TOX2 detection in advanced imaging applications:

• Confocal microscopy: Biotin-conjugated antibodies often provide superior sensitivity for detecting nuclear transcription factors like TOX2 in fixed tissues

• Super-resolution microscopy: Direct fluorophore conjugates may be preferred for techniques requiring precise localization (STORM, PALM), while structured illumination microscopy (SIM) can accommodate biotin-streptavidin detection systems

• Intravital imaging: Direct conjugates typically perform better due to reduced tissue penetration issues and fewer binding steps

• FRET applications: Direct conjugates are strongly preferred due to precise distance requirements

The choice between biotin-conjugated TOX2 antibody and direct fluorophore conjugates should be guided by the specific experimental requirements, with particular consideration of target abundance, required sensitivity, and the technical limitations of the imaging platform .