TUBB9 Antibody

What is the TUBB9 antibody and what does it target?

TUBB9 antibody (also referenced as TuBB-9 in some literature) is a monoclonal antibody that targets the nuclear protein Ki-67, which is a cellular marker for proliferation . This antibody is primarily used in research settings to study cell proliferation patterns and has been effectively employed in combination with fluorescence microscopy techniques to visualize Ki-67 expression in cellular nuclei. The antibody has been validated for applications including immunofluorescence microscopy, particularly after conjugation with fluorescent molecules like FITC.

What are the primary applications of TUBB9 antibody in research?

TUBB9 antibody is utilized in several research applications, including:

• Immunofluorescence microscopy after fluorescent labeling (particularly with FITC)

• Analysis of cell proliferation through Ki-67 detection

• Photochemical internalization studies investigating targeted cellular delivery

• Visualization of nuclear protein expression during cell cycle progression

• Combination with other antibodies in multiplexed immunostaining to evaluate cellular states

Research has demonstrated the utility of TUBB9 in photochemical internalization studies where cells are first incubated with compounds like Fimaporfin before antibody application .

What considerations should be made for proper TUBB9 antibody storage and handling?

While specific storage information for TUBB9 antibody isn't detailed in the provided sources, general antibody handling principles apply:

• Store antibody aliquots at recommended temperatures (typically -20°C for long-term storage)

• Avoid repeated freeze-thaw cycles by preparing working aliquots

• When working with fluorescently labeled TUBB9 (e.g., TUBB9-FITC conjugates), protect from light exposure

• Maintain proper pH conditions (pH ~7.5 for most applications, but protocol-specific conditions may vary)

• Follow manufacturer recommendations for reconstitution and dilution buffers

For experimental applications, TUBB9 antibody has been successfully used in protocols involving incubation periods of approximately 4 hours at specified concentrations .

What controls should be included when designing experiments with TUBB9 antibody?

Proper experimental design with TUBB9 antibody should include:

• Negative controls:

  • Isotype controls (matched antibody class but irrelevant specificity)

  • Secondary antibody only (for indirect detection methods)

  • Cells known to lack Ki-67 expression

• Positive controls:

  • Cell lines with verified Ki-67 expression (such as proliferating cancer cell lines)

  • Tissues with known patterns of Ki-67 expression

• Validation controls:

  • Comparison with other validated Ki-67 antibodies

  • Correlation with other proliferation markers

These controls are essential for accurate interpretation of staining patterns and verification of antibody specificity.

What is the optimal protocol for FITC labeling of TUBB9 antibody?

Based on research methodologies, the following protocol has been established for FITC labeling of TUBB9 antibody :

• Antibody Preparation:

  • Dilute TUBB9 antibody 1:5 in sodium carbonate buffer (160 nM Na₂CO₃ and 333 nM NaHCO₃, pH 9.3)

  • Centrifuge at 1855× g for 20 minutes

  • Collect residues in the filter using buffer solution (pH 9.3)

• Conjugation:

  • Add 1 mg/mL of FITC (dissolved in DMSO) to the TUBB9 solution

  • Incubate at room temperature with constant agitation for 2 hours

• Purification:

  • Use a Sephadex column (e.g., NAP-25) pre-buffered with tris-buffered saline (TBS, pH 8.2)

  • Centrifuge the eluate

  • Rinse the filter twice with 500 μL TBS (pH 7.5) and add to the sample

• Verification:

  • Determine the final concentration of antibody and fluorescent dye using absorption spectrum analysis

This methodology has been successfully employed in studies involving photochemical internalization with Fimaporfin, where cells were incubated with TUBB9-FITC construct for 4 hours .

How can researchers troubleshoot low signal or high background issues when using TUBB9 antibody?

When encountering signal issues with TUBB9 antibody:

For Low Signal:

• Verify antibody activity and concentration

• Increase antibody concentration or incubation time

• Optimize antigen retrieval methods if applicable

• Ensure target accessibility (permeabilization for intracellular targets)

• Check detection system sensitivity (for fluorescent detection, ensure appropriate filters and exposure settings)

For High Background:

• Increase blocking duration or concentration (using appropriate blocking agents)

• Optimize washing steps (increase number or duration)

• Decrease antibody concentration

• Pre-absorb antibody with potential cross-reactive proteins

• For fluorescent applications, ensure proper signal thresholding to suppress false positives

Researchers have found that optimizing signal-to-noise thresholds is critical when analyzing dense MS spectra, with some studies suggesting a signal-to-noise threshold of ≥7 may be appropriate for antibody analysis rather than standard values of 3 .

What considerations should be made when using TUBB9 antibody for targeted cellular delivery?

For targeted cellular delivery applications using TUBB9 antibody:

• Internalization Dynamics:

  • TUBB9 antibody has been successfully used in photochemical internalization studies, where cells are first incubated with the photosensitizer (e.g., Fimaporfin for 18h) and then with TUBB9-FITC for 4h

  • After washing, cells can be exposed to specific wavelength light (420 nm has been used) at defined energy levels (0.25 J/cm²) to trigger endosomal disruption and antibody release

• Validation Approaches:

  • Fluorescence microscopy at multiple timepoints (pre-irradiation, 30 min post-irradiation, and 24h post-irradiation) to track antibody localization and release from endosomes

  • Comparison of internalization efficiency between different cell types

  • Co-localization studies with endosomal markers

• Optimization Parameters:

  • Cell type-specific incubation times

  • Concentration of photosensitizer and antibody

  • Light exposure parameters (wavelength, intensity, duration)

  • Buffer composition for optimal internalization

This approach allows for temporal and spatial control of TUBB9 antibody delivery for studying nuclear protein dynamics.

How can researchers analyze Ki-67 expression patterns using TUBB9 antibody across different cell types?

Analysis of Ki-67 expression using TUBB9 antibody requires:

• Standardized Quantification Methods:

  • Percentage of positive cells (labeling index)

  • Intensity scoring (negative, weak, moderate, strong)

  • Digital image analysis with appropriate thresholding

  • Correlation with cell cycle markers

• Experimental Design Considerations:

  • Consistent fixation and processing methods across samples

  • Matched exposure settings for image acquisition

  • Inclusion of proliferative and quiescent control cell populations

  • Technical replicates to account for staining variability

• Advanced Analysis Approaches:

  • Single-cell analysis of Ki-67 expression in heterogeneous populations

  • Correlation with other proliferation markers (e.g., PCNA, MCM proteins)

  • Spatial analysis of Ki-67 distribution within the nucleus

  • Temporal dynamics during cell cycle progression

A systematic approach using these considerations enables reliable cross-comparison of Ki-67 expression between different experimental conditions and cell types.

What strategies can be employed to validate TUBB9 antibody specificity?

Validating TUBB9 antibody specificity requires multiple complementary approaches:

• Analytical Validation:

  • Western blotting to confirm target molecular weight

  • Immunoprecipitation followed by mass spectrometry

  • Peptide competition assays

  • Immunodepletion studies

• Biological Validation:

  • Correlation of staining with known proliferation states

  • Comparison with other validated Ki-67 antibodies

  • Testing in cells with genetic Ki-67 knockdown/knockout

  • Evaluation across multiple cell lines and tissue types

• Advanced Specificity Testing:

  • Epitope mapping to determine precise binding region

  • Cross-reactivity assessment with related proteins

  • Testing in various species if cross-reactivity is expected

  • Analysis under different experimental conditions (fixation methods, buffer compositions)

Validation is particularly important when developing new experimental applications for TUBB9 antibody beyond established protocols.

How can Design of Experiments (DOE) approach be applied to optimize TUBB9 antibody-based assays?

Design of Experiments methodology provides a systematic framework for optimizing TUBB9 antibody protocols:

• Key Factors to Consider:

  • Antibody concentration

  • Incubation time and temperature

  • Buffer composition (pH, ionic strength)

  • Blocking conditions

  • Detection system parameters

• Experimental Design Structure:

  • Full factorial or fractional factorial designs to assess factor interactions

  • Response surface methodology to identify optimal conditions

  • Plackett-Burman designs for screening many factors

  • Central composite designs for optimization studies

• Implementation Strategy:

  Factor	Low Level	High Level	Control Range (±)
  Antibody Conc.	1 μg/mL	10 μg/mL	1 μg/mL
  Temperature	4°C	37°C	2°C
  pH	6.8	7.8	0.2
  Incubation Time	1 h	18 h	0.5 h

• Analysis Approaches:

  • Statistical software (like MODDE) for analyzing factorial experiments

  • Response optimization for multiple output variables

  • Establishment of a "design space" for robust assay performance

Researchers have successfully applied DOE approaches to antibody development processes, making it a valuable methodology for TUBB9 antibody optimization .

What are the critical considerations when using TUBB9 antibody in multiplexed imaging?

Multiplexed imaging with TUBB9 antibody requires careful consideration of:

• Antibody Compatibility:

  • Species origin compatibility between antibodies

  • Isotype selection to avoid cross-reactivity

  • Epitope accessibility when multiple targets are proximal

• Signal Separation:

  • Spectral separation between fluorophores

  • Sequential staining protocols when needed

  • Appropriate controls for spectral overlap/bleed-through

• Advanced Multiplexing Strategies:

  • Sequential bleaching and restaining approaches

  • Cyclic immunofluorescence methods

  • Mass cytometry or imaging mass cytometry for high-dimensional analysis

  • Antibody stripping and reprobing protocols

• Validation Requirements:

  • Single-stain controls for each antibody

  • Fluorescence minus one (FMO) controls

  • Signal correlation analysis between sequential imaging rounds

  • Comparison with single-plex staining results

These considerations enable researchers to effectively use TUBB9 antibody in complex multiplexed imaging experiments while maintaining specificity and sensitivity.

How can researchers use TUBB9 antibody in conjunction with emerging technologies for advanced cellular analysis?

Integration of TUBB9 antibody with emerging technologies offers new research possibilities:

• TUBB9 in Super-Resolution Microscopy:

  • Optimization of fluorophore selection for STORM, PALM, or STED microscopy

  • Sample preparation considerations for nanoscale resolution

  • Quantitative analysis of Ki-67 distribution at sub-diffraction resolution

• Single-Cell Analysis Applications:

  • Combination with single-cell RNA sequencing for correlative analysis

  • Integration with mass cytometry for high-dimensional phenotyping

  • Microfluidic approaches for temporal analysis of Ki-67 dynamics

• Live Cell Imaging Considerations:

  • Fragment-based antibody approaches for improved penetration

  • Optimization of non-perturbative labeling strategies

  • Photoactivatable antibody conjugates for spatiotemporal control

• Computational Analysis Integration:

  • Machine learning algorithms for pattern recognition in TUBB9 staining

  • Automated image analysis workflows for high-throughput screening

  • Multi-parametric data integration frameworks

These approaches extend the utility of TUBB9 antibody beyond traditional applications, enabling more sophisticated analysis of Ki-67 expression and cellular proliferation dynamics.

How should researchers interpret contradictory results with TUBB9 antibody across different experimental platforms?

When faced with contradictory results:

• Systematic Verification:

  • Validate antibody lot consistency and activity

  • Compare fixation and sample preparation methods

  • Evaluate detection system sensitivity differences

  • Consider epitope accessibility variations between platforms

• Resolution Approaches:

  • Use multiple antibody clones targeting different Ki-67 epitopes

  • Implement orthogonal methods for proliferation assessment

  • Perform dose-response studies to identify threshold effects

  • Consider cell type-specific differences in Ki-67 expression

• Analytical Considerations:

  • Standardize quantification methods across platforms

  • Account for differences in detection limit and dynamic range

  • Apply appropriate statistical methods for cross-platform normalization

  • Document all experimental variables that might influence results

Research has shown that apparent contradictions in antibody studies often stem from methodological differences rather than true biological variations .

What are the most common artifacts when using TUBB9 antibody and how can they be distinguished from true signals?

Common artifacts include:

• Edge Effects:

  • Identified by: Increased signal at tissue or cell margins

  • Mitigation: Optimize fixation, improve washing techniques, adjust imaging settings

• Autofluorescence:

  • Identified by: Signal in negative controls, broad spectral emission

  • Mitigation: Use appropriate quenching methods, spectral unmixing, narrow bandpass filters

• Non-specific Binding:

  • Identified by: Diffuse cytoplasmic staining, signal in negative control samples

  • Mitigation: Optimize blocking, use validated diluents, include appropriate controls

• Fixation Artifacts:

  • Identified by: Inconsistent staining patterns correlated with fixation time/method

  • Mitigation: Standardize fixation protocols, use positive control tissues

• Cross-reactivity:

  • Identified by: Unexpected subcellular localization, staining in tissues without Ki-67

  • Mitigation: Validate with multiple antibodies, perform blocking studies

Creating a systematic artifact atlas specific to TUBB9 antibody applications can help researchers distinguish true biological signals from technical artifacts.

How can researchers effectively report TUBB9 antibody methodologies in publications to ensure reproducibility?

Best practices for reporting TUBB9 antibody methods include:

• Antibody Documentation:

  • Complete antibody identification (clone, isotype, supplier, catalog number, lot number)

  • Validation methods used to confirm specificity

  • Concentration or dilution used

• Protocol Details:

  • Complete buffer compositions with exact pH values

  • Precise timing of each step

  • Temperature conditions

  • Detailed antigen retrieval methods if applicable

• Controls Description:

  • Full details of positive and negative controls

  • Images of control experiments

  • Criteria used to establish positive staining

• Image Acquisition Parameters:

  • Microscope specifications

  • Camera/detector settings

  • Exposure times

  • Image processing methods with software versions

• Quantification Methods:

  • Detailed analysis workflow

  • Software tools used for quantification

  • Thresholding criteria

  • Statistical methods for data analysis

Following these reporting standards enhances reproducibility across different research laboratories.

How might TUBB9 antibody be used in combination with emerging antibody technologies?

Emerging combinations include:

• Bispecific Antibody Approaches:

  • Development of bispecific constructs combining TUBB9 with antibodies targeting other proliferation markers

  • Engineering antibodies that simultaneously target Ki-67 and deliver therapeutic payloads

  • Creation of multi-functional antibodies that can stimulate T cell immunity while binding to Ki-67

• Engineered Antibody Formats:

  • Single-domain antibodies for improved tissue penetration

  • pH-sensitive antibody conjugates for controlled release

  • Split antibody complementation systems for proximity detection

• Novel Conjugation Strategies:

  • Site-specific conjugation methods for improved homogeneity

  • Cleavable linkers for controlled release of payloads

  • Environmentally responsive linkers for targeted delivery

These advanced approaches expand the utility of TUBB9 beyond traditional immunostaining applications toward therapeutic and diagnostic innovations.

What role might TUBB9 antibody play in understanding the relationship between cell proliferation and disease processes?

TUBB9 antibody can contribute to understanding disease mechanisms through:

• Cancer Research Applications:

  • Correlation of Ki-67 expression patterns with tumor progression

  • Identification of proliferative heterogeneity within tumors

  • Evaluation of treatment response based on Ki-67 dynamics

• Neurodegenerative Disease Studies:

  • Assessment of neuronal proliferation in response to injury

  • Evaluation of stem cell proliferation in neurodegenerative models

  • Correlation of aberrant cell cycle reentry with pathology

• Immunological Investigations:

  • Tracking lymphocyte proliferation during immune responses

  • Monitoring proliferative exhaustion in chronic infections

  • Evaluating proliferative responses to immunotherapy

• Regenerative Medicine:

  • Assessment of stem cell proliferation during tissue repair

  • Evaluation of proliferative capacity in engineered tissues

  • Correlation of regenerative outcomes with proliferation patterns

By providing detailed information about cell proliferation states, TUBB9 antibody enables researchers to establish mechanistic links between proliferation abnormalities and disease processes.

How can researchers integrate computational approaches with TUBB9 antibody data for more comprehensive analysis?

Advanced computational integration includes:

• Image Analysis Automation:

  • Deep learning for Ki-67 positive cell identification

  • Convolutional neural networks for pattern recognition

  • Automated quantification across large tissue sections

• Multi-omics Integration:

  • Correlation of Ki-67 expression with transcriptomic profiles

  • Integration with proteomic data to identify co-expressed markers

  • Pathway analysis incorporating Ki-67 status

• Spatial Analysis:

  • Neighborhood analysis of Ki-67 positive cells

  • Spatial statistics to identify proliferative hotspots

  • Tissue microenvironment characterization based on proliferation patterns

• Predictive Modeling:

  • Development of prognostic models incorporating Ki-67 data

  • Simulation of proliferative responses to therapeutic interventions

  • Virtual tissue modeling incorporating proliferation dynamics

These computational approaches transform descriptive TUBB9 antibody data into predictive models with greater biological insight.