TPX2 Antibody, Biotin conjugated

What is TPX2 and what cellular functions should researchers consider when designing experiments with TPX2 antibodies?

TPX2 functions as a multifunctional protein with several critical roles in cell division. When planning experiments with TPX2 antibodies, researchers should consider its roles as:

• A spindle assembly factor required for normal assembly of mitotic spindles

• A mediator of microtubule assembly during apoptosis

• A facilitator of chromatin and/or kinetochore-dependent microtubule nucleation

• A mediator of Aurora A kinase (AURKA) localization to spindle microtubules

• An activator of AURKA by promoting autophosphorylation at Thr-288 and protecting this residue against dephosphorylation

TPX2 is regulated through inactivation upon binding to importin-alpha. At mitosis onset, GOLGA2 interacts with importin-alpha, liberating TPX2 and allowing it to activate AURKA kinase and stimulate local microtubule nucleation . Experimental design must account for these cell cycle-dependent interactions and appropriate timing.

How does a biotin-conjugated TPX2 antibody differ methodologically from unconjugated antibodies in experimental applications?

Biotin-conjugated TPX2 antibodies offer distinct methodological advantages over unconjugated formats:

Detection sensitivity:

• The biotin-streptavidin system provides significant signal amplification due to the high affinity binding

• Multiple detection options through various streptavidin conjugates (fluorophores, enzymes, quantum dots)

Experimental flexibility:

• Compatible with multiple detection methods without requiring species-specific secondary antibodies

• Enables multi-color immunofluorescence with antibodies from the same host species

• Facilitates protein isolation through streptavidin-coated beads or columns

Methodological considerations:

• Requires blocking of endogenous biotin (especially in tissues with high biotin content)

• May increase background if the biotinylation level is too high

• Potential steric hindrance if biotin conjugation affects the antibody binding site

When using biotin-conjugated TPX2 antibodies, researchers should implement blocking controls, isotype controls, and preabsorption with recombinant TPX2 protein to distinguish specific signals from background.

What are the validated applications for TPX2 antibodies and what methodological adaptations are needed for each?

Based on available data, TPX2 antibodies have been validated for several applications requiring specific methodological adaptations:

Western Blot (WB):

• Expected molecular weight: 100 kDa (corresponding to p100 isoform)

• Sample preparation: Complete cell lysis is crucial for adequate TPX2 detection

• Reducing conditions required

• Recommended blocking: 5% non-fat milk or BSA in TBST

Immunohistochemistry (IHC):

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0)

• Nuclear localization expected with occasional cytoplasmic staining

• Consider counterstaining with hematoxylin for contrast

Immunocytochemistry/Immunofluorescence (ICC/IF):

• Fixation: 4% paraformaldehyde (10 min) followed by permeabilization with 0.1% Triton X-100

• Cell cycle-dependent localization: diffuse nuclear in interphase, spindle-associated in mitosis

• Recommended co-staining with tubulin to visualize spindle structures

Immunoprecipitation (IP):

• Lysis buffer recommendation: 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate with protease inhibitors

• Pre-clearing recommended to reduce background

For biotin-conjugated antibodies, additional methodological adaptations include avidin/biotin blocking steps and adjustment of detection systems to utilize streptavidin conjugates.

How can TPX2 antibodies be used to study the interaction between TPX2 and Aurora A kinase?

Investigating the TPX2-Aurora A interaction requires specialized methodological approaches:

Co-immunoprecipitation optimization:

• Use mild lysis buffers to preserve protein-protein interactions

• Include phosphatase inhibitors to maintain phosphorylation status of Thr-288 on Aurora A

• Consider crosslinking approaches to stabilize transient interactions

• Compare results using both TPX2 and Aurora A antibodies for precipitation

Proximity ligation assay (PLA) protocol:

• Fix cells at specific cell cycle stages (particularly early mitosis)

• Use validated TPX2 and Aurora A antibodies from different species

• Follow with species-specific PLA probes

• Quantify interaction signals in different cell compartments

Biochemical mapping with domain-specific antibodies:
TPX2 activates Aurora A by promoting its autophosphorylation at Thr-288 and protects this residue against dephosphorylation . This interaction can be mapped using antibodies targeting specific domains:

What controls and validation steps are essential when using TPX2 antibodies for immunofluorescence analysis of spindle structures?

Proper controls and validation steps are crucial for accurate immunofluorescence analysis of TPX2 in spindle structures:

Essential controls:

• Peptide competition assay: Pre-incubation of the antibody with immunizing peptide should abolish specific staining

• siRNA knockdown validation: Cells treated with TPX2 siRNA should show reduced staining intensity

• Cell cycle markers: Co-staining with cyclin B1 or phospho-histone H3 to identify mitotic cells

• Spindle marker controls: Co-staining with α-tubulin to confirm spindle localization pattern

Validation protocol steps:

• Compare staining patterns between multiple antibodies targeting different TPX2 epitopes

• Verify specificity through western blot correlation with immunofluorescence intensity

• Confirm expected cell cycle-dependent localization changes

• Validate through ectopic expression of tagged TPX2 constructs

Quantitative analysis approaches:

• Measure TPX2-to-tubulin fluorescence intensity ratio along spindle microtubules

• Quantify co-localization coefficients with Aurora A kinase

• Map TPX2 distribution relative to chromatin and kinetochores

How can biotin-conjugated TPX2 antibodies be used to investigate microtubule branching nucleation?

Investigating TPX2's role in microtubule branching nucleation requires specialized experimental approaches:

In vitro reconstitution assay:
TPX2 plays a key role in branching microtubule nucleation together with augmin, γTuRC, and XMAP215 . For studying this process:

• Domain-specific inhibition:

  • Use antibodies targeting specific TPX2 domains to block interactions

  • Data shows at least three successive domains are necessary for significant microtubule binding in vitro

• Visualization strategy:

  • Employ TIRF microscopy for real-time observation

  • Use biotin-conjugated TPX2 antibodies with streptavidin-conjugated quantum dots for tracking

Xenopus egg extract experiments:
Research has shown that TPX2's C-terminal half can induce branched, fan-like microtubule structures, but this requires augmin . When designing experiments:

• Depletion-add back approach:

  • Immunodeplete endogenous TPX2

  • Add back full-length or domain-specific TPX2 constructs

  • Use TPX2 antibodies to confirm depletion efficiency

• Quantification methods:

  • Measure branching angle distribution

  • Count number of branches per microtubule length

  • Analyze microtubule network density

• Critical controls:

  • Augmin depletion (prevents branching regardless of TPX2 presence)

  • Addition of importin-α (should inhibit TPX2 activity)

  • RanQ69L addition (releases TPX2 from importin-α inhibition)

How can TPX2 antibodies be employed in studying the effects of TPX2 inhibition on cancer cell sensitivity to microtubule-targeting drugs?

TPX2 antibodies provide powerful tools for investigating TPX2's role in cancer therapeutic responses:

Experimental design for drug synergy studies:
Research has shown that knockdown of TPX2 sensitized pancreatic cancer cells to paclitaxel treatment, with paclitaxel dose response curves shifting left when combined with TPX2 siRNAs . To investigate this:

• Validation of TPX2 inhibition:

  • Use antibodies to confirm knockdown efficiency at protein level

  • Establish baseline TPX2 expression across cell line panels

  • Correlate expression with baseline sensitivity to microtubule-targeting drugs

• Cell viability assay protocols:

  • Plate cells at optimal density (typically 3000-5000 cells/well)

  • Transfect with TPX2 siRNA, then treat with paclitaxel 6 hours later

  • Determine cell viability using SRB or MTT assay after 96 hours

  • Generate dose-response curves with and without TPX2 knockdown

• Mechanistic investigation techniques:

  • Analyze mitotic arrest via flow cytometry

  • Assess apoptosis through Cell Death ELISA assay

  • Visualize spindle abnormalities using immunofluorescence

Key findings to validate:

• TPX2 knockdown alone reduces cell viability in pancreatic cancer cells

• Combined treatment with TPX2 siRNA and paclitaxel shows synergistic effects

• The effect appears specific to anti-mitotic agents, as similar experiments with gemcitabine did not show synergistic effects

What are the critical storage and handling parameters for maintaining TPX2 antibody functionality?

Maintaining TPX2 antibody functionality requires attention to several critical storage and handling parameters:

Storage conditions:

• Temperature: Store at -20°C for long-term storage

• Formulation: Typically in PBS with 40% glycerol and 0.05% sodium azide as preservative

• Aliquoting: Prepare small, single-use aliquots to avoid repeated freeze-thaw cycles

Stability parameters:

Critical handling considerations:

• Avoid repeated freeze-thaw cycles (limit to <5 total cycles)

• When thawing, allow antibody to reach room temperature before opening to prevent condensation

• Mix gently by inversion or finger-tapping; avoid vortexing which can denature antibodies

• For biotin-conjugated antibodies, protect from light during handling

Working solution preparation:

• Include 0.01% sodium azide in working solutions for extended use

• Consider adding carrier protein (0.1-0.5% BSA) to dilute antibody solutions

• For biotin-conjugated antibodies, prepare fresh working dilutions before each experiment

What are the potential cross-reactivity issues with TPX2 antibodies and how can they be addressed?

Addressing potential cross-reactivity when using TPX2 antibodies requires systematic validation approaches:

Common cross-reactivity concerns:

• TPX2 shares sequence homology with other microtubule-associated proteins

• Multiple isoforms and splice variants may be recognized differently

• Post-translational modifications may affect epitope accessibility

Validation strategies:

• Test antibody reactivity in TPX2-knockout or knockdown samples

• Perform peptide competition assays with immunizing peptide

• Compare staining patterns between antibodies targeting different TPX2 epitopes

• Validate with recombinant TPX2 protein as positive control

Species cross-reactivity considerations:
The immunogen used for many TPX2 antibodies corresponds to synthetic peptides within human TPX2 amino acids 150-200 . While human reactivity is well-established, cross-reactivity with other species should be validated experimentally.

What fixation and antigen retrieval methods optimize TPX2 detection in cell and tissue samples?

Optimizing TPX2 detection requires careful consideration of fixation and antigen retrieval methods:

Cell samples:

Tissue samples:

For optimal results with TPX2 antibodies in immunohistochemical applications, heat-induced epitope retrieval in citrate buffer (pH 6.0) has proven effective in tissue microarrays of pancreatic tumors .

How can TPX2 antibodies be utilized for studying TPX2 as a potential therapeutic target in pancreatic cancer?

Investigation of TPX2 as a cancer therapeutic target using antibodies requires specialized methodological approaches:

TPX2 expression analysis in clinical samples:
Research has shown that TPX2 expression is upregulated in pancreatic cancer cell lines at both mRNA and protein levels compared to normal cells, and immunohistochemical staining showed higher TPX2 expression in pancreatic tumors compared to normal tissue .

• Tissue microarray optimization:

  • Standardize antigen retrieval (citrate buffer, pH 6.0)

  • Optimize antibody concentration using positive/negative controls

  • Use digital pathology for quantitative scoring

• Expression correlation analysis:

  • Compare TPX2 levels with clinical outcomes

  • Correlate with known markers (Ki-67, Aurora A)

  • Stratify by tumor subtype and stage

Functional studies:
TPX2 knockdown using siRNAs effectively reduced pancreatic cancer cell growth in tissue culture, induced apoptosis, and inhibited growth in soft agar and in nude mice .

• Cell death quantification methods:

  • Cell Death ELISA for histone-DNA fragmentation assessment

  • Analyze concentration-dependent effects of TPX2 inhibition

  • Compare with established apoptosis inducers

• Growth inhibition assays:

  • Colony formation in soft agar showed significant reduction with TPX2 knockdown

  • Nude mouse xenograft growth was inhibited by TPX2 siRNA treatment

  • These effects validate TPX2 as a potential therapeutic target

• Combination therapy approaches:

  • TPX2 knockdown sensitized pancreatic cancer cells to paclitaxel treatment

  • Combined treatment showed synergistic effects on cell viability

  • This suggests potential for TPX2-targeted therapies in combination with taxanes

What methodological approaches allow investigation of TPX2 domain-specific functions using antibodies?

Investigating domain-specific functions of TPX2 requires sophisticated methodological approaches:

Domain mapping strategies:
TPX2 contains several functional domains with distinct roles. Research has shown that multiple domains in TPX2 cooperatively mediate microtubule binding, with at least three successive domains necessary for significant microtubule binding in vitro .

• Epitope-specific antibody selection:

  • Choose antibodies targeting specific TPX2 domains:

    • N-terminal (aa 1-43): Aurora A activation

    • Middle region (aa 150-200): Importin-α binding

    • C-terminal regions: Microtubule binding

• Domain blocking experiments:

  • Pre-incubate recombinant TPX2 with domain-specific antibodies

  • Measure effects on protein interactions and functions

Microtubule nucleation activity analysis:
TPX2's C-terminal half can induce branched, fan-like microtubule structures in Xenopus egg extract, but this requires augmin .

• Domain contribution assay:

  • Use domain-specific antibodies to block specific functions

  • Measure effects on microtubule nucleation

  • Quantify branching frequency and microtubule density

• Quantitative parameters: