TUBA4A Antibody, Biotin conjugated

What is TUBA4A and why is it significant in cytoskeletal research?

TUBA4A (Tubulin Alpha 4A) is a major constituent of microtubules, which are cylindrical structures consisting of laterally associated linear protofilaments composed of alpha- and beta-tubulin heterodimers. Microtubules grow by the addition of GTP-tubulin dimers to their ends, forming a stabilizing cap. Below this cap, tubulin dimers exist in GDP-bound state due to the GTPase activity of alpha-tubulin .

TUBA4A is also known by several aliases including TUBA1, Alpha-tubulin 1, Testis-specific alpha-tubulin, Tubulin H2-alpha, and Tubulin alpha-1 chain . As a critical cytoskeletal component, TUBA4A plays essential roles in:

• Maintaining cell structure and shape

• Facilitating intracellular transport

• Enabling cell division through mitotic spindle formation

• Supporting cell motility

Researchers focus on TUBA4A because alterations in microtubule dynamics are implicated in various pathological conditions, making it both a potential biomarker and therapeutic target.

What are the structural characteristics of biotin-conjugated TUBA4A antibodies?

Biotin-conjugated TUBA4A antibodies consist of:

• An antibody component (either monoclonal or polyclonal) that specifically targets TUBA4A epitopes

• A biotin molecule covalently attached to the antibody

These antibodies are available in different formats:

The biotin conjugation allows for enhanced detection sensitivity through the strong interaction between biotin and avidin/streptavidin conjugates in secondary detection systems .

How do biotin-conjugated TUBA4A antibodies function in detection systems?

Biotin-conjugated TUBA4A antibodies function through a multi-step process:

• The antibody portion binds specifically to TUBA4A protein in the sample

• The biotin portion serves as a high-affinity binding site for avidin/streptavidin conjugates

• Detection reagents (typically avidin/streptavidin conjugated to enzymes like HRP or fluorophores) bind to the biotin

• Signal generation occurs through enzymatic reactions or fluorescence

In ELISA applications, this typically follows this process:

• Samples containing TUBA4A are added to wells pre-coated with capture antibody

• Biotin-conjugated TUBA4A antibody is added and binds to captured TUBA4A

• Avidin-HRP is added and binds to biotin

• TMB substrate is added, producing color change in proportion to TUBA4A concentration

This approach offers amplified signal detection due to the multiple binding sites on avidin/streptavidin for biotin, enhancing sensitivity.

What are the validated applications for biotin-conjugated TUBA4A antibodies?

Biotin-conjugated TUBA4A antibodies have been validated for multiple experimental applications:

When selecting an application, researchers should consider:

• The specific question being addressed

• Sample type and preparation method

• Required sensitivity and specificity

• Available detection systems

What is the optimal protocol for using biotin-conjugated TUBA4A antibodies in Western blotting?

For optimal Western blot results with biotin-conjugated TUBA4A antibodies:

Sample Preparation:

• Prepare cell/tissue lysates in appropriate lysis buffer

• Determine protein concentration (Bradford/BCA assay)

• Mix samples with loading buffer and denature at 95°C for 5 minutes

• Load 20-50 μg protein per lane

Gel Electrophoresis and Transfer:

• Separate proteins on 10-12% SDS-PAGE gel

• Transfer to PVDF/nitrocellulose membrane

Immunoblotting:

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

• Incubate with biotin-conjugated TUBA4A antibody at manufacturer-recommended dilution (typically 1:1000-1:8000)

• Wash 3-5 times with TBST

• Incubate with streptavidin-HRP (1:2000-1:5000) for 1 hour at room temperature

• Wash 3-5 times with TBST

• Develop using ECL substrate

• Image using appropriate detection system

Expected Results:

• TUBA4A should appear at approximately 50 kDa

• Multiple cell lines can be used as positive controls, including A431, HeLa, and HEK-293T cells

Western blots using TUBA4A antibodies have been successfully performed on multiple cell and tissue lysates including 293, A431, A549, HeLa, Jurkat, mouse brain, and rat brain samples .

How should biotin-conjugated TUBA4A antibodies be used in ELISA applications?

For ELISA applications using biotin-conjugated TUBA4A antibodies:

Sandwich ELISA Protocol:

• Coat microplate wells with capture antibody specific to TUBA4A

• Block with appropriate blocking buffer

• Add samples or standards containing TUBA4A

• Add biotin-conjugated TUBA4A antibody (follow manufacturer's recommended dilution)

• Add avidin/streptavidin-HRP conjugate

• Add TMB substrate solution

• Add stop solution (typically sulfuric acid)

• Measure absorbance at 450nm ± 10nm

Key Considerations:

• The standard curve should be prepared using recombinant TUBA4A

• Include appropriate positive and negative controls

• For quantitative analysis, construct a standard curve by plotting mean absorbance for each standard versus TUBA4A concentration

• Sample concentrations can be determined by comparing absorbance values to the standard curve

This method enables detection of native, not recombinant, TUBA4A in tissue homogenates, cell lysates, and other biological fluids .

What factors affect the specificity and sensitivity of biotin-conjugated TUBA4A antibodies?

Several factors can influence the performance of biotin-conjugated TUBA4A antibodies:

Antibody Characteristics:

• Clonality: Monoclonal antibodies typically offer higher specificity but may have lower sensitivity compared to polyclonal antibodies

• Host species: Rabbit-derived antibodies often provide higher affinity and specificity for human TUBA4A

• Target epitope: Antibodies targeting different regions (C-terminal vs. other domains) may have varying specificity profiles

Sample-Related Factors:

• Sample preparation: Incomplete protein denaturation or inappropriate lysis buffers may affect antibody binding

• Post-translational modifications: These may mask epitopes or alter antibody recognition

• Expression levels: Low TUBA4A expression may require antibodies with higher sensitivity or signal amplification methods

Experimental Conditions:

• Antibody concentration: Using optimal dilutions is critical (1:1000-1:8000 for WB, varies for other applications)

• Incubation conditions: Temperature, duration, and buffer composition affect binding kinetics

• Blocking efficiency: Insufficient blocking leads to high background; excessive blocking may mask epitopes

Researchers should validate antibody performance for their specific experimental system and optimize conditions accordingly.

How can researchers troubleshoot non-specific binding with biotin-conjugated TUBA4A antibodies?

When encountering non-specific binding with biotin-conjugated TUBA4A antibodies:

Common Problems and Solutions:

Positive Control Recommendations:

• Always include known positive controls such as A431, HeLa, or HEK-293T cell lysates

• For human samples, multiple cell lines show consistent TUBA4A expression (Jurkat, K562, U937)

• For cross-species studies, validated reactivity has been demonstrated in mouse, rat, rabbit, chicken, and zebrafish samples

How can biotin-conjugated TUBA4A antibodies be integrated into multiplexed detection systems?

Biotin-conjugated TUBA4A antibodies can be leveraged in multiplexed detection systems through several advanced approaches:

Multicolor Immunofluorescence:

• Use biotin-conjugated TUBA4A antibody alongside directly-labeled antibodies against other targets

• Detect TUBA4A using streptavidin conjugated to a unique fluorophore (e.g., Alexa Fluor 488)

• Analyze co-localization with other proteins within the same sample

Multiplex Western Blotting:

• Combine biotin-conjugated TUBA4A antibody with directly-conjugated antibodies of different hosts/isotypes

• Use distinct detection systems (streptavidin-HRP for biotin, species-specific secondary antibodies for others)

• Detect using fluorescent secondary antibodies with different emission spectra

ELISA-Based Multiplex Arrays:

• Incorporate biotin-conjugated TUBA4A antibodies into multiplexed bead arrays

• Detect multiple analytes simultaneously using spectrally distinct streptavidin conjugates

• Analyze using appropriate flow cytometry or imaging platforms

These approaches enable researchers to investigate TUBA4A in relation to other proteins within the same sample, providing insights into protein interactions and co-regulatory mechanisms.

What are the considerations for using biotin-conjugated TUBA4A antibodies in studying microtubule dynamics?

When using biotin-conjugated TUBA4A antibodies to study microtubule dynamics:

Technical Considerations:

• Sample Fixation: Preserve microtubule structure using paraformaldehyde fixation (typically 4% for 15-20 minutes)

• Permeabilization: Use gentle detergents (0.1-0.2% Triton X-100) to maintain microtubule integrity

• Co-staining: Combine with markers for microtubule post-translational modifications (acetylation, tyrosination)

• Imaging: Use high-resolution microscopy (confocal or super-resolution) to visualize microtubule networks

Experimental Approaches:

• Microtubule Stability Assays:

  • Treat cells with microtubule-stabilizing (taxol) or destabilizing (nocodazole) agents

  • Use biotin-conjugated TUBA4A antibodies to visualize changes in microtubule organization

• Cell Cycle Analysis:

  • Synchronize cells at different cell cycle stages

  • Analyze TUBA4A distribution during mitosis, particularly at the mitotic spindle

• Drug Response Studies:

  • Evaluate microtubule dynamics in response to therapeutic compounds

  • Correlate structural changes with functional outcomes

Data Interpretation:

• Consider that antibodies may preferentially detect stable versus dynamic microtubule populations

• Recognize that TUBA4A is one of several α-tubulin isoforms that may have overlapping distributions

• Validate findings with complementary approaches (live-cell imaging with fluorescent tubulin)

What approaches can be used to study post-translational modifications of TUBA4A using biotin-conjugated antibodies?

Studying post-translational modifications (PTMs) of TUBA4A requires specialized approaches:

Experimental Strategies:

• Two-dimensional Western blotting:

  • First dimension: Separate by isoelectric point

  • Second dimension: Separate by molecular weight

  • Detect with biotin-conjugated TUBA4A antibody

  • PTMs appear as shifts in isoelectric point or molecular weight

• Sequential Immunoprecipitation:

  • First IP: Use antibodies against specific PTMs (acetylation, phosphorylation)

  • Second detection: Probe with biotin-conjugated TUBA4A antibody

  • Alternatively, IP with TUBA4A antibody and probe with PTM-specific antibodies

• Mass Spectrometry Analysis:

  • Immunoprecipitate TUBA4A using biotin-conjugated antibodies

  • Analyze by mass spectrometry to identify PTMs

  • Quantify modification stoichiometry

Common TUBA4A Modifications to Investigate:

• C-terminal tyrosination/detyrosination

• Polyglutamylation (characteristic of neuronal microtubules)

• Acetylation (marker of stable microtubules)

• Phosphorylation (often associated with cell cycle regulation)

Data Interpretation Considerations:

• Compare modification patterns across different cell types or conditions

• Correlate modifications with microtubule stability and function

• Consider that specific PTMs may affect antibody recognition

How should researchers quantitatively analyze TUBA4A expression data?

For rigorous quantitative analysis of TUBA4A expression:

Western Blot Quantification:

• Use digital image analysis software (ImageJ, Image Studio, etc.)

• Define regions of interest around TUBA4A bands

• Measure integrated density or area under curve

• Normalize to appropriate loading controls:

  • Housekeeping proteins (GAPDH, β-actin)

  • Total protein measurement (Ponceau S, REVERT stain)

• Express results as relative TUBA4A expression compared to control conditions

ELISA Quantification:

• Generate standard curve using recombinant TUBA4A (4-parameter logistic regression)

• Ensure samples fall within the linear range of the standard curve

• Calculate TUBA4A concentrations based on absorbance values

• Normalize to total protein concentration

• Perform replicate measurements (minimum triplicate) for statistical analysis

Statistical Analysis:

• For comparing two groups: t-test (paired or unpaired)

• For multiple groups: ANOVA with appropriate post-hoc tests

• For non-normally distributed data: non-parametric alternatives (Mann-Whitney, Kruskal-Wallis)

• Report mean ± standard deviation/SEM with appropriate significance levels

Visualization:

• Present data using bar graphs or box plots

• Include individual data points to show distribution

• Clearly indicate sample size and statistical significance

How should researchers interpret differences in TUBA4A detection across different experimental platforms?

When encountering differences in TUBA4A detection across platforms:

Common Discrepancies and Interpretations:

Resolution Strategies:

• Technical Validation:

  • Repeat experiments with multiple technical replicates

  • Use alternative antibody clones targeting different TUBA4A epitopes

  • Compare results with non-antibody methods (RT-PCR, mass spectrometry)

• Biological Interpretation:

  • Consider post-translational modifications affecting epitope recognition

  • Evaluate potential isoform-specific detection differences

  • Assess context-dependent protein interactions masking epitopes

• Data Integration:

  • Develop weighted scoring systems incorporating multiple detection methods

  • Focus on consistent trends rather than absolute values

  • Use orthogonal approaches to validate key findings

How can biotin-conjugated TUBA4A antibodies contribute to research on neurodegenerative disorders?

TUBA4A has emerging significance in neurodegenerative research:

Current Knowledge and Applications:

• TUBA4A mutations have been identified in familial amyotrophic lateral sclerosis (ALS)

• Microtubule dynamics are altered in various neurodegenerative conditions

• Biotin-conjugated TUBA4A antibodies can help characterize:

  • Expression changes in disease models

  • Altered microtubule stability and organization

  • Interactions with other disease-associated proteins

Methodological Approaches:

• Tissue Analysis:

  • Compare TUBA4A expression and localization in patient versus control brain tissues

  • Evaluate co-localization with pathological inclusions

• Cellular Models:

  • Analyze TUBA4A dynamics in neurons derived from patient iPSCs

  • Study effects of TUBA4A mutations on microtubule stability and transport

• Animal Models:

  • Track TUBA4A expression changes in disease progression

  • Test therapeutic interventions targeting microtubule stability

These approaches provide insight into how TUBA4A alterations contribute to neurodegeneration and identify potential therapeutic targets for conditions like ALS.

What emerging technologies might enhance the utility of biotin-conjugated TUBA4A antibodies?

Several emerging technologies show promise for expanding TUBA4A research:

Advanced Imaging Techniques:

• Super-resolution microscopy (STORM, PALM) for nanoscale analysis of TUBA4A organization

• Expansion microscopy to physically enlarge samples for improved resolution

• Live-cell imaging combined with biotin-based labeling strategies

Single-Cell Analysis:

• Mass cytometry (CyTOF) incorporating biotin-conjugated TUBA4A antibodies

• Single-cell Western blotting for heterogeneity analysis

• Spatial transcriptomics combined with TUBA4A protein detection

Proximity Labeling Approaches:

• BioID or APEX2 fusions with TUBA4A to identify proximal interacting proteins

• Integration with biotin-conjugated antibodies for validation studies

Computational Methods:

• Machine learning algorithms for automated microtubule network analysis

• Integrative multi-omics approaches incorporating TUBA4A proteomic data

• Simulation of microtubule dynamics based on experimental data

These technologies will enable researchers to gain deeper insights into TUBA4A biology, from molecular interactions to whole-cell functions, advancing our understanding of cytoskeletal dynamics in health and disease.