AURKA Antibody

What is AURKA and what cellular functions does it regulate?

AURKA (Aurora Kinase A), also known as STK6, STK15, or AIK, belongs to the Ser/Thr protein kinase family. This protein plays a critical role in cell cycle regulation during anaphase and/or telophase by participating in microtubule formation and stabilization. AURKA regulates mitosis through its association with centrosomes in vivo . It is highly expressed in testis and weakly in skeletal muscle, thymus, and spleen . Recent evidence indicates AURKA may be implicated in tumor development and progression, making it a significant target for cancer research .

What is the expected molecular weight of AURKA in Western blot applications?

AURKA has a calculated molecular weight of approximately 46 kDa, which is consistent with its observed molecular weight in SDS-PAGE applications . Different antibodies report slightly different observed weights:

• Proteintech antibody 10297-1-AP: 46 kDa

• Proteintech antibody 28371-1-AP: 45-50 kDa

• Invitrogen Monoclonal Antibody MA5-15803: approximately 48 kDa

The protein typically migrates as a 46 kDa band in SDS-PAGE applications, though slight variations may occur depending on post-translational modifications or experimental conditions .

What cell lines are recommended as positive controls for AURKA antibody validation?

Based on the search results, the following cell lines have been validated as positive controls for AURKA antibody testing:

HeLa cells appear to be the most consistently used positive control across multiple antibodies and should be considered a primary choice for initial validation studies .

What are the recommended applications and dilutions for AURKA antibodies?

Different AURKA antibodies have been optimized for specific applications with recommended dilutions:

For optimal results, it is recommended to titrate each antibody in your specific testing system as performance may vary depending on sample type and experimental conditions .

What antigen retrieval methods are effective for AURKA immunohistochemistry?

For optimal immunohistochemical detection of AURKA:

• Proteintech 10297-1-AP: Suggested antigen retrieval with TE buffer pH 9.0; alternatively, citrate buffer pH 6.0 may be used

• Boster Bio A00246-3: Heat-mediated antigen retrieval using EDTA buffer (pH 8.0) is recommended for paraffin-embedded tissue sections

• For enzyme antigen retrieval in immunocytochemistry: IHC enzyme antigen retrieval reagent (such as AR0022) with 15-minute incubation has been effective for Caco-2 cells

The choice of antigen retrieval method should be optimized based on tissue type and fixation protocols to maximize specific signal while minimizing background.

What are the optimal storage conditions for preserving AURKA antibody activity?

Most AURKA antibodies require similar storage conditions:

• Storage temperature: -20°C for long-term storage

• Stability: Generally stable for one year after shipment when properly stored

• For reconstituted lyophilized antibodies: Can be stored at 4°C for one month or aliquoted and stored at -20°C for six months

• Repeated freeze-thaw cycles should be avoided

• Some preparations contain glycerol (typically 50%) and sodium azide (0.02%) in PBS at pH 7.3

• Smaller (20μl) sizes may contain 0.1% BSA

Proper aliquoting upon receipt is recommended for antibodies that will be used multiple times to prevent degradation from freeze-thaw cycles.

How is AURKA expression altered in cancer tissues compared to normal tissues?

Nuclear staining for AURKA is typically weak or nonexistent in normal tissue but strong in tumor tissue . AURKA is overexpressed in many human cancers, including breast, ovarian, and colorectal cancers . This differential expression pattern makes AURKA a potential biomarker for cancer detection and a target for anticancer drug development.

In experimental models:

• Positive IHC for AURKA has been detected in human breast cancer tissue

• Paraffin-embedded sections of human liver cancer tissue and Hashimoto thyroiditis tissue have shown positive AURKA staining

• The overexpression pattern correlates with AURKA's role in tumor development and progression

Researchers should consider using paired normal and tumor tissue samples when designing experiments to evaluate AURKA expression changes in cancer contexts.

How can AURKA antibodies be used to evaluate the efficacy of AURKA inhibitors in cancer models?

AURKA antibodies are valuable tools for assessing the efficacy of AURKA inhibitors:

• Monitoring target engagement: AURKA antibodies can detect changes in total AURKA protein levels or subcellular localization following inhibitor treatment

• Evaluating downstream effects: The research by Katsha et al. (2017) demonstrated that AURKA can regulate EIF4E and cap-dependent translation in upper gastrointestinal adenocarcinomas, with inhibition by alisertib reversing these molecular events

• Studying resistance mechanisms: AURKA inhibitor MLN8237 was found to upregulate PD-L1 expression in breast cancer cells via STAT3 phosphorylation, potentially explaining poor clinical outcomes with MLN8237 monotherapy

• Combination therapy assessment: AURKA antibodies were instrumental in demonstrating that combining MLN8237 with anti-PD-L1 antibody improved therapeutic outcomes in colon cancer models by enhancing immune cell infiltration

• Immunophenotyping: Flow cytometry with AURKA antibodies can quantify changes in AURKA expression at the cellular level following inhibitor treatment

These approaches allow researchers to comprehensively evaluate both direct effects on AURKA and downstream consequences of AURKA inhibition.

What is the AURKA-EIF4E-c-MYC axis and how can it be studied using AURKA antibodies?

The AURKA-EIF4E-c-MYC axis represents a novel oncogenic pathway identified by Katsha et al. (2017), where AURKA activates EIF4E (eukaryotic translation initiation factor 4E) leading to increased cap-dependent translation and upregulation of c-MYC in cancer cells .

Key aspects of this pathway that can be studied using AURKA antibodies include:

• Mechanistic studies: AURKA was found to bind to and inactivate protein phosphatase 2A (PP2A), a negative regulator of EIF4E, leading to EIF4E phosphorylation and activation in an AKT-, ERK1/2-, and mTOR-independent manner

• Resistance to mTOR inhibitors: AURKA overexpression was detected in everolimus-resistant upper gastrointestinal cancer models, with AURKA inhibition resensitizing these cells to treatment

• Therapeutic targeting: The AURKA-EIF4E-c-MYC axis represents a novel therapeutic target, particularly for everolimus-resistant tumors or cancers that show overexpression of AURKA and activation of EIF4E and c-MYC

Researchers can use AURKA antibodies in combination with antibodies against phosphorylated EIF4E and c-MYC to monitor this signaling axis in various experimental settings, including in vitro cell models and tumor xenografts.

Are there known cross-reactivity issues with AURKA antibodies?

Cross-reactivity can be a significant concern with AURKA antibodies. The search results revealed a specific case study examining cross-reactivity between N6AMT1 antibodies and AURKA :

• An N6AMT1 antibody (referred to as antibody IV) raised against the N-terminal part of the protein (residues 115-214) produced multiple non-specific bands in immunoblot analysis but did not recognize AURKA recombinant protein with high affinity

• Conversely, when testing whether AURKA antibodies might recognize N6AMT1, researchers found that a mouse monoclonal AURKA antibody (sc-56881) did not recognize N6AMT1 (no visible band at 23 kDa) nor recombinant EGFP-tagged N6AMT1 protein

This information indicates that:

• Cross-reactivity between AURKA and N6AMT1 appears to be minimal with the tested antibodies

• Researchers should still validate antibody specificity in their experimental systems

• Knockout or knockdown controls are valuable for confirming antibody specificity

What validation methods are most effective for confirming AURKA antibody specificity?

Based on the search results, several methods have been employed to validate AURKA antibody specificity:

• Knockout/knockdown validation: Two publications cited using AURKA knockdown to validate antibody specificity for antibody 10297-1-AP

• Recombinant protein testing: Testing antibodies against purified recombinant AURKA protein, as demonstrated in the cross-reactivity studies with N6AMT1

• Multiple antibody validation: Using multiple antibodies targeting different epitopes of AURKA to confirm consistent results

• Size verification: Confirming that the detected protein band corresponds to the expected molecular weight (approximately 46 kDa)

• Positive control cell lines: Using cell lines known to express AURKA, such as HeLa, HEK-293, and cancer cell lines

For comprehensive validation, researchers should combine multiple approaches, with knockout/knockdown controls providing the most definitive evidence of specificity.

What are the common challenges in immunofluorescence applications with AURKA antibodies?

Immunofluorescence applications with AURKA antibodies can present several challenges:

• Background signal: Multiple antibodies, including A00246-3, may require careful optimization to minimize non-specific staining

• Fixation and permeabilization: For intracellular AURKA detection in flow cytometry, cells need to be fixed with 4% paraformaldehyde and permeabilized with appropriate buffer to allow antibody access

• Cell cycle-dependent expression: AURKA expression and localization change throughout the cell cycle, which can affect interpretation of results if cell populations are not synchronized

• Antibody selection for co-staining: When performing multi-label immunofluorescence, care must be taken to select compatible primary and secondary antibodies to avoid cross-reactivity

• Optimal dilution ranges: Different antibodies require specific dilution ranges for optimal signal-to-noise ratio in IF/ICC applications:

  • 10297-1-AP: 1:200-1:800

  • 28371-1-AP: 1:200-1:800

  • A00246-3: 5 μg/mL for Caco-2 cells

Optimization strategies should include titration of primary and secondary antibodies, testing different fixation and permeabilization protocols, and including proper controls (positive, negative, and secondary-only).

How can AURKA antibodies be used to study AURKA's role in immune checkpoint regulation?

Recent research has revealed an unexpected connection between AURKA inhibition and immune checkpoint regulation, which can be studied using AURKA antibodies:

• PD-L1 upregulation: Research by Hu et al. (2023) demonstrated that the AURKA inhibitor MLN8237 upregulated PD-L1 expression in breast cancer cells in a time- and concentration-dependent manner

• Mechanism of action: This PD-L1 upregulation was associated with increased phosphorylation of STAT3. AURKA antibodies can be used alongside phospho-STAT3 antibodies to study this regulatory connection

• T-cell infiltration: MLN8237 treatment decreased CD3+ and CD8+ T cell infiltration in 4T1-breast tumor xenograft models. AURKA antibodies, combined with immune cell markers, can help monitor these changes

• Combination therapy approaches: The findings suggest that combining AURKA inhibitors with immune checkpoint inhibitors (anti-PD-L1 antibody) could overcome resistance and improve therapeutic outcomes in cancer treatment

• Potential experimental designs:

  • Flow cytometry using AURKA and PD-L1 antibodies to quantify expression changes

  • Immunohistochemistry to assess AURKA, PD-L1, and T cell marker expression in tumor sections

  • Western blotting to analyze AURKA/STAT3/PD-L1 signaling pathways

This emerging area represents an important intersection between targeted therapy and immunotherapy approaches in cancer treatment.

What are the considerations for using AURKA antibodies in studying its role in treatment resistance?

AURKA overexpression has been implicated in resistance to various cancer therapies, and AURKA antibodies are valuable tools for studying these resistance mechanisms:

• mTOR inhibitor resistance: Katsha et al. (2017) detected overexpression of endogenous AURKA in everolimus-resistant upper gastrointestinal cancer cell models. AURKA antibodies can be used to monitor AURKA expression levels in resistant vs. sensitive cells

• Mechanism of resistance: AURKA was found to mediate phosphorylation of EIF4E, activation of cap-dependent translation, and increase in c-MYC protein levels in an AKT-, ERK1/2-, and mTOR-independent manner, contributing to treatment resistance

• Therapeutic targeting: Targeting AURKA using genetic knockdown or the small-molecule inhibitor alisertib reversed resistance mechanisms and decreased cancer cell survival in both acquired and intrinsic resistant cell models

• Immune evasion: AURKA inhibitor MLN8237 was found to upregulate PD-L1, potentially allowing tumor cells to evade immune surveillance when cell cycle progression is disrupted, suggesting a complex relationship between AURKA inhibition and treatment response

• Experimental approaches:

  • Western blotting with AURKA antibodies to compare expression levels between sensitive and resistant cells

  • Immunofluorescence to assess AURKA localization changes in resistant cells

  • Co-immunoprecipitation using AURKA antibodies to identify novel interaction partners in resistant cells

Understanding these resistance mechanisms can inform the development of more effective combination therapies and biomarker strategies for patient selection.

How can AURKA phosphorylation states be monitored, and what is their significance?

Although the search results don't specifically mention phospho-specific AURKA antibodies, they do indicate that AURKA phosphorylation state is important in its function and regulation:

• Functional activation: As a serine/threonine kinase, AURKA's activity is regulated through phosphorylation events. Different phosphorylation states may correlate with different functional outcomes

• Downstream signaling: AURKA was shown to mediate phosphorylation of EIF4E in cancer cells, suggesting that AURKA's own phosphorylation state might influence its ability to phosphorylate substrates

• Inhibitor response: Changes in AURKA phosphorylation status following inhibitor treatment could serve as a biomarker for target engagement and efficacy

• Experimental approaches:

  • Phospho-specific antibodies (if available) could be used to distinguish between different activation states of AURKA

  • Phosphatase treatment controls in Western blotting can help identify bands representing phosphorylated forms of AURKA

  • Phos-tag gels can separate different phosphorylated forms of AURKA for subsequent detection with total AURKA antibodies

• Technical considerations:

  • Sample preparation methods should preserve phosphorylation status (phosphatase inhibitors in lysis buffers)

  • Quick sample processing at cold temperatures helps maintain phosphorylation states

  • Quantitative comparisons of phosphorylated vs. total AURKA can provide insights into activation status

Although phospho-specific AURKA antibodies weren't explicitly mentioned in the search results, researchers interested in AURKA phosphorylation should inquire with antibody manufacturers about the availability of such specialized reagents.