AURKA Antibody, FITC conjugated

What is AURKA and why is it a significant research target?

AURKA (Aurora Kinase A) is a serine/threonine kinase belonging to the Ser/Thr protein kinase family that plays a critical role in cell cycle regulation during anaphase and telophase by influencing microtubule formation and stabilization . It has emerged as a significant research target due to its overexpression in numerous human cancers including breast, ovarian, and colorectal malignancies . AURKA's involvement in tumor development and progression has positioned it as a potential target for anticancer drug development, making antibodies against this protein valuable tools for understanding cancer biology and developing therapeutic strategies .

What applications are AURKA antibodies typically used for in research settings?

AURKA antibodies are versatile research tools employed across multiple experimental techniques. According to validation data, these antibodies can be utilized in:

FITC-conjugated variants are particularly valuable for applications requiring direct fluorescent detection, eliminating the need for secondary antibodies in immunofluorescence-based techniques .

How should AURKA antibodies be stored to maintain optimal activity?

For maximum shelf life and activity preservation, AURKA antibodies, including FITC-conjugated variants, should be stored at -20°C to -80°C according to manufacturer specifications . The antibody is typically supplied in a stabilizing buffer containing preservatives such as 0.03% Proclin 300 and 50% glycerol in PBS (pH 7.4) to maintain protein integrity during storage . Repeated freeze-thaw cycles should be avoided as they can compromise antibody functionality through protein denaturation and aggregation . For routine use, small working aliquots may be prepared to minimize these detrimental effects, particularly for fluorophore-conjugated antibodies where the fluorescent tag may be sensitive to repeated temperature fluctuations.

What controls should be included when using AURKA antibodies in experimental protocols?

When designing experiments with AURKA antibodies, researchers should incorporate several critical controls:

• Positive controls: Utilize samples known to express AURKA, such as HeLa cells or human breast cancer tissue, which have been validated for positive detection .

• Negative controls: Include samples where AURKA expression is absent or reduced, or omit the primary antibody while maintaining all other experimental conditions.

• Isotype controls: Employ non-specific antibodies of the same isotype (e.g., rabbit IgG for polyclonal rabbit anti-AURKA antibodies) to assess potential background binding .

• Knockdown/knockout validation: For definitive specificity confirmation, comparison with AURKA-depleted samples using siRNA knockdown or CRISPR-Cas9 knockout approaches provides robust validation of antibody specificity .

How can AURKA antibodies be utilized to investigate radioresistance mechanisms in cancer cells?

AURKA antibodies serve as essential tools for investigating radioresistance mechanisms in cancer, particularly through the AURKA/NFκB axis. Research demonstrates that cervical squamous carcinoma cells develop radioresistance through AURKA overexpression, which can be monitored using immunofluorescence techniques with FITC-conjugated antibodies .

For investigating this phenomenon, researchers should:

• Establish radioresistant sublines by subjecting parental cancer cell lines (e.g., SiHa cervical carcinoma cells) to fractionated X-irradiation doses, isolating resistant populations at defined radiation thresholds (e.g., 40Gy) .

• Compare AURKA and phosphorylated AURKA (pAURKA) expression between parental and radioresistant sublines using immunofluorescence with FITC-conjugated antibodies, enabling quantitative analysis of expression differences.

• Employ secondary validation through complementary techniques such as western blotting to confirm elevation of AURKA protein levels in radioresistant cells.

• Integrate functional studies through AURKA inhibition or knockdown experiments to establish causative relationships between AURKA expression and radioresistance phenotypes .

What methodological approaches can optimize AURKA detection in formalin-fixed paraffin-embedded (FFPE) cancer tissues?

Optimizing AURKA detection in FFPE tissue specimens requires careful attention to antigen retrieval and antibody validation:

• Antigen retrieval optimization: Two primary buffer systems have shown efficacy for AURKA detection:

  • Sodium citrate buffer (10 mM, pH 6.0) as employed in lung cancer tissue studies

  • TE buffer (pH 9.0) with alternative citrate buffer (pH 6.0) options for breast cancer tissue specimens

• Signal amplification strategy: Implement the streptavidin-HRP detection system for chromogenic visualization or direct FITC-conjugated antibodies for fluorescence microscopy .

• Counterstaining approach: For chromogenic detection, hematoxylin provides effective nuclear counterstaining, while DAPI is preferred for fluorescence-based methods .

• Quantification methodology: Employ semi-quantitative H-score systems with independent investigator assessment to ensure reliable quantification of AURKA expression patterns across tissue specimens .

How can AURKA antibodies be integrated into studies investigating combinatorial cancer treatment approaches?

AURKA antibodies provide crucial tools for investigating the efficacy of combinatorial cancer treatment regimens, particularly when evaluating AURKA inhibition alongside radiation or platinum-based chemotherapies:

• Experimental design for in vitro studies should incorporate:

  • Cell viability assays comparing single-agent vs. combination treatments

  • Immunofluorescence using FITC-conjugated AURKA antibodies to monitor expression changes following treatment

  • Mechanistic investigations of DNA damage through γH2AX foci quantification

  • Apoptosis and senescence measurements to characterize cell death mechanisms

• In vivo xenograft experimental approaches should include:

  • Doxycycline-inducible AURKA knockdown systems paired with FITC-conjugated antibodies for expression validation

  • Comparative tumor growth assessments between control, single-agent, and combination treatment groups

  • Immunohistochemical analysis of excised tumors to evaluate treatment effects on AURKA expression and downstream pathways

• Immune checkpoint modulation analysis:

  • Investigation of relationships between AURKA inhibition and immune checkpoint expression (PD-L1, B7-H3) using co-staining approaches with FITC-conjugated AURKA antibodies and complementary immune checkpoint detection reagents

What strategies can minimize photobleaching when working with FITC-conjugated AURKA antibodies?

FITC-conjugated antibodies are susceptible to photobleaching, which can compromise experimental outcomes, particularly in quantitative imaging applications. Researchers should implement these methodological considerations:

• Sample preparation optimization:

  • Incorporate anti-fade mounting media containing photoprotective agents

  • Store prepared slides in light-protected containers at 4°C

  • Minimize exposure to ambient light during all procedural steps

• Microscopy parameters adjustment:

  • Utilize low-intensity excitation light and short exposure times

  • Employ neutral density filters to reduce illumination intensity

  • Capture reference images first, followed by experimental samples

• Alternative approaches for critical experiments:

  • Consider more photostable fluorophores (Alexa Fluor conjugates) for extended imaging sessions

  • Implement computational correction algorithms for photobleaching compensation during image analysis

How should cross-reactivity concerns be addressed when employing AURKA antibodies across different species?

Cross-reactivity considerations are crucial when applying AURKA antibodies across experimental models. The available data indicates:

When working across species boundaries:

• Validate antibody specificity in each species through western blotting to confirm correct molecular weight detection (expected ~46-48 kDa) .

• Perform sequence homology analysis between human AURKA (UniProt ID: O14965) and target species to predict potential cross-reactivity.

• For unvalidated species applications, conduct preliminary titration experiments across concentration ranges to optimize signal-to-noise ratios.

• Consider epitope mapping information when available - antibodies targeting highly conserved regions offer greater cross-species utility .

What are the most common sources of false-positive signals when using FITC-conjugated AURKA antibodies, and how can they be mitigated?

False-positive signals can significantly compromise experimental integrity when working with FITC-conjugated AURKA antibodies. Common sources and mitigation strategies include:

• Autofluorescence from biological samples:

  • Implement tissue autofluorescence quenching using Sudan Black B (0.1-0.3%) treatment prior to antibody incubation

  • Utilize spectral unmixing during image acquisition to distinguish antibody signal from autofluorescence

  • Include unstained control samples for background signal subtraction during analysis

• Non-specific binding:

  • Optimize blocking protocols using species-appropriate serum (5-10%) or BSA (3-5%)

  • Include isotype control experiments using FITC-conjugated non-specific rabbit IgG at equivalent concentrations

  • Implement additional washing steps with detergent-containing buffers to reduce background

• Spectral overlap with other fluorophores:

  • Design multicolor panels with appropriate spectral separation

  • Perform single-color controls for compensation when using flow cytometry

  • Utilize sequential scanning approaches during confocal microscopy

How should researchers interpret AURKA expression levels in the context of different cancer models?

Interpreting AURKA expression requires consideration of cancer-specific contexts and methodological standardization:

• Expression baseline establishment:

  • Compare expression levels against matched normal tissues rather than cell lines alone

  • Implement semi-quantitative scoring systems such as H-score for IHC or mean fluorescence intensity for flow cytometry

  • Correlate expression levels with clinical parameters when using patient-derived samples

• Cancer-specific considerations:

  • In breast cancer: AURKA antibody detection frequently reveals overexpression, particularly in breast cancer tissue compared to normal breast epithelium

  • In lung cancer: AURKA expression correlates with response to platinum-based therapies and radiation, necessitating pre-treatment expression analysis

  • In cervical cancer: Radioresistant phenotypes demonstrate elevated AURKA/pAURKA expression compared to radiosensitive counterparts

• Subcellular localization analysis:

  • Evaluate both cytoplasmic and nuclear AURKA localization patterns

  • Consider cell cycle phase-dependent expression when interpreting results

  • Correlate with phosphorylation status using phospho-specific antibodies where applicable

How can FITC-conjugated AURKA antibodies facilitate investigation of AURKA inhibitors as sensitizers for conventional cancer therapies?

FITC-conjugated AURKA antibodies provide valuable tools for investigating AURKA inhibitors as sensitizing agents for conventional cancer therapies:

• Mechanistic investigation approaches:

  • Utilize FITC-conjugated antibodies to quantify changes in AURKA expression/localization following inhibitor treatment

  • Implement high-content imaging to correlate AURKA levels with DNA damage markers in single-cell analysis

  • Develop live-cell imaging protocols using cell-permeable AURKA activity sensors alongside fixed-cell AURKA antibody staining

• Combination therapy optimization:

  • Determine optimal scheduling of AURKA inhibition relative to radiation or chemotherapy through time-course analysis of AURKA expression and activity

  • Evaluate impact of AURKA inhibition on radiation-induced DNA damage repair through γH2AX co-staining experiments

  • Investigate potential synergistic mechanisms through pathway analysis targeting NFκB and downstream effectors

• Predictive biomarker development:

  • Correlate baseline AURKA expression (detected via FITC-conjugated antibodies) with treatment response outcomes

  • Develop standardized protocols for AURKA detection in clinical specimens to inform patient stratification strategies

  • Integrate AURKA expression analysis into multi-parameter predictive models for therapy response

What considerations should guide experimental design when investigating relationships between AURKA and immune checkpoints?

Recent evidence indicates complex relationships between AURKA inhibition and immune checkpoint expression, requiring careful experimental design considerations:

• Multiparameter analysis approaches:

  • Implement co-staining protocols with FITC-conjugated AURKA antibodies and complementary immune checkpoint antibodies (PD-L1, B7-H3)

  • Utilize multicolor flow cytometry to quantify correlations between AURKA and immune checkpoint expression at single-cell resolution

  • Develop multiplex immunofluorescence protocols for spatial relationship analysis in tissue specimens

• Temporal dynamics investigation:

  • Design time-course experiments to determine sequence of expression changes following AURKA inhibition

  • Evaluate acute versus chronic AURKA inhibition effects on immune checkpoint expression

  • Correlate changes with functional immune response assays when investigating immunotherapy combinations

• Translational research considerations:

  • Analyze archival patient samples with paired AURKA and immune checkpoint staining

  • Develop tissue microarray approaches for high-throughput screening across tumor types

  • Integrate findings with genomic and transcriptomic data to identify mechanistic pathways connecting AURKA activity and immune evasion