HAUS3 Antibody

What is HAUS3 and what role does it play in cellular processes?

HAUS3, also known as C4orf15, is a component of the HAUS augmin-like complex which regulates mitotic spindle assembly and centrosome integrity and is required for completion of cytokinesis. The complex interacts with the gamma-tubulin ring complex, and this interaction is essential for proper spindle assembly during cell division . HAUS3 contributes to maintaining centrosome integrity and proper mitotic progression, making it an important target for cell division studies .

What is the molecular weight of HAUS3 and how is it typically observed in experimental settings?

While the calculated molecular weight of HAUS3 is approximately 70 kDa, it is typically observed at 62-65 kDa in experimental conditions such as Western blot analysis . This discrepancy between calculated and observed molecular weights is important to consider when designing experiments and interpreting results. The difference may be attributed to post-translational modifications, protein folding characteristics, or alternative splicing that affect gel migration patterns.

Where does HAUS3 localize within cells during different cell cycle phases?

HAUS3 demonstrates specific subcellular localization patterns associated with its function in mitosis. The protein localizes to interphase centrosomes and to mitotic spindle microtubules during cell division . This distinct localization pattern is consistent with its role in spindle assembly and centrosome function, making it an excellent marker for studying centrosome dynamics and mitotic progression in various research contexts.

What types of HAUS3 antibodies are available and how should researchers select the appropriate antibody?

Several types of HAUS3 antibodies are available for research applications, including:

• Polyclonal antibodies targeting different epitopes (e.g., AA 1-100, AA 138-187, AA 291-340, AA 372-400, AA 379-408, AA 384-603)

• Unconjugated antibodies for standard applications

• Conjugated antibodies (AbBy Fluor® 594, Biotin) for specialized detection methods

When selecting an antibody, researchers should consider:

• The specific epitope required for their application

• The species reactivity needed (human, mouse, rat)

• The intended application (WB, IHC, IF, ELISA, FACS)

• Whether a conjugated or unconjugated antibody is more suitable for the detection system

How can researchers validate the specificity of HAUS3 antibodies?

Validating HAUS3 antibody specificity requires multiple approaches:

• Testing in positive control cell lines with known HAUS3 expression (HepG2 and HeLa cells have been confirmed to express HAUS3)

• Performing knockdown/knockout experiments as negative controls

• Confirming the correct molecular weight band (62-65 kDa) in Western blots

• Verifying expected subcellular localization pattern (centrosomes and spindle microtubules) using immunofluorescence

• Cross-validating results using multiple antibodies targeting different epitopes of HAUS3

What are the recommended reactivity and cross-reactivity considerations for HAUS3 antibodies?

Most commercial HAUS3 antibodies demonstrate reactivity with human samples, with some showing cross-reactivity with mouse and rat models . When working with non-human species, it's essential to:

• Verify sequence homology between human HAUS3 and the target species

• Perform preliminary validation experiments with appropriate controls

• Consider that antibodies targeting different epitopes may show different cross-reactivity profiles

• Note that an antibody showing reactivity with human, guinea pig, and bat samples has been reported for specific epitopes (AA 138-187)

What are the optimal parameters for detecting HAUS3 by Western blotting?

For successful Western blot detection of HAUS3:

For optimal results, researchers should:

• Include protease inhibitors in lysis buffers to prevent degradation

• Optimize transfer conditions for the relatively large protein

• Consider longer blocking and antibody incubation times for enhanced specificity

What protocol is recommended for immunofluorescence detection of HAUS3?

For immunofluorescence applications:

• Fix cells using 4% paraformaldehyde (10-15 minutes at room temperature)

• Permeabilize with 0.2% Triton X-100 in PBS (10 minutes)

• Block with 5% BSA or normal serum (1 hour)

• Incubate with primary HAUS3 antibody at 1:20-1:200 dilution

• Use appropriate fluorophore-conjugated secondary antibody or direct-conjugated primary (like AbBy Fluor® 594)

• Co-stain with centrosome/microtubule markers and DNA dyes for proper localization analysis

• Analyze using confocal or super-resolution microscopy

For enhanced visualization of centrosomal structures, consider:

• Co-staining with γ-tubulin as a centrosome marker

• Using cell cycle synchronization to enrich for mitotic cells

• Implementing z-stack imaging to capture the full three-dimensional structure

What are the recommended parameters for immunohistochemical detection of HAUS3?

For immunohistochemistry applications:

For optimal IHC results:

• Test both suggested antigen retrieval methods to determine optimal conditions

• Include appropriate positive and negative controls in each experimental run

• Consider slide-to-slide variability when interpreting results

• Optimize antibody incubation time and temperature based on signal strength

How can HAUS3 antibodies be used to study spindle assembly mechanisms?

HAUS3 antibodies offer valuable tools for investigating spindle assembly mechanisms:

• Use immunofluorescence to track HAUS3 localization throughout mitotic progression

• Perform co-immunoprecipitation experiments to identify HAUS3 interaction partners during spindle assembly

• Combine with tubulin staining to analyze microtubule organization and density in normal versus compromised conditions

• Implement live-cell imaging with fluorescently tagged HAUS3 to monitor dynamic recruitment to spindle structures

• Compare HAUS3 localization patterns in cells treated with microtubule-stabilizing or destabilizing agents

In these experiments, researchers should focus on the role of HAUS3 in mediating interactions between centrosomes and spindle microtubules, particularly through its association with the gamma-tubulin ring complex.

What strategies can be employed to study HAUS3's role in centrosome integrity?

To investigate HAUS3's contribution to centrosome integrity:

• Use super-resolution microscopy with HAUS3 antibodies to examine detailed centrosome structure

• Perform HAUS3 knockdown/knockout studies and analyze resulting centrosome abnormalities

• Examine HAUS3 recruitment during centrosome duplication using synchronized cell populations

• Investigate potential phosphorylation or other post-translational modifications of HAUS3 during centrosome cycle

• Study HAUS3 localization and function in cells with centrosome amplification

These approaches can provide insights into how HAUS3 contributes to maintaining proper centrosome structure and function throughout the cell cycle.

How can researchers investigate HAUS3's interactions with the gamma-tubulin ring complex?

To study the critical interaction between HAUS3 and the gamma-tubulin ring complex:

• Perform co-immunoprecipitation using HAUS3 antibodies followed by immunoblotting for gamma-tubulin components

• Conduct proximity ligation assays to visualize in situ protein-protein interactions

• Use fluorescence resonance energy transfer (FRET) to detect direct molecular interactions

• Implement structured illumination microscopy to visualize co-localization at the nanoscale level

• Compare interaction dynamics during different cell cycle phases and under various perturbation conditions

Understanding this interaction is crucial as it represents a key mechanism by which HAUS3 contributes to proper spindle assembly during mitosis.

How should researchers address unexpected bands or patterns when using HAUS3 antibodies?

When encountering unexpected results:

For multiple Western blot bands:

• Consider potential alternative splicing or isoforms of HAUS3

• Evaluate whether bands represent post-translationally modified forms

• Test for potential degradation products by modifying sample preparation

• Validate specific bands using siRNA knockdown

• Compare results across multiple antibodies targeting different epitopes

For atypical immunofluorescence patterns:

• Verify fixation and permeabilization conditions are appropriate

• Compare with literature-reported localization patterns

• Use co-staining with established centrosome/spindle markers

• Test different cell types to rule out cell-specific variations

• Optimize antibody concentration to reduce non-specific binding

What controls should be included when designing experiments with HAUS3 antibodies?

Proper experimental design requires appropriate controls:

• Positive controls: HepG2 or HeLa cells for Western blot and immunofluorescence applications

• Negative controls: Primary antibody omission, isotype controls, and HAUS3-depleted samples

• Specificity controls: Competing peptide blocking or pre-absorption tests

• Cross-reactivity controls: Testing in species with predicted reactivity (mouse, rat)

• Application-specific controls: Loading controls for Western blot, tissue-specific positive controls for IHC, and co-staining controls for IF

How can researchers optimize HAUS3 antibody performance for challenging applications?

For challenging detection scenarios:

• Test different epitope-targeting antibodies if one region proves difficult to detect

• Consider alternative sample preparation methods for better antigen preservation

• Implement signal amplification systems for low-abundance detection

• Test both N-terminal and C-terminal targeting antibodies to ensure complete detection

• Optimize blocking conditions to reduce background in high-sensitivity applications

• Consider specialized fixation protocols for preserving delicate centrosome structures

How can HAUS3 antibodies be utilized in cancer research?

HAUS3 antibodies offer valuable tools for cancer research:

• Compare HAUS3 expression between normal and malignant tissues using IHC and Western blot

• Correlate expression with markers of chromosomal instability and centrosome amplification

• Examine HAUS3 localization in cancer cells with abnormal mitotic spindles

• Investigate potential correlations between HAUS3 expression/localization and cancer progression

• Study the effects of anti-mitotic drugs on HAUS3 function in cancer cells

• Analyze HAUS3 expression in human liver cancer tissue, which has been validated as a positive control

What considerations are important when studying HAUS3 in developmental and stem cell research?

For developmental and stem cell applications:

• Select antibodies validated in appropriate developmental model systems

• Compare HAUS3 expression and localization between stem cells and differentiated cells

• Analyze HAUS3 function during symmetric versus asymmetric cell divisions

• Investigate potential roles in centrosome inheritance during development

• Consider epitope accessibility, which may vary in different developmental contexts

• Optimize fixation conditions carefully, as embryonic tissues often require specialized protocols

How can researchers apply HAUS3 antibodies to study neurodegenerative diseases?

For neurodegenerative disease research:

• Examine HAUS3 expression in neural cells with centrosome-related defects

• Investigate potential alterations in mitotic spindle assembly in disease models

• Study HAUS3 interactions with disease-associated proteins

• Analyze centrosome structure in patient-derived samples

• Explore connections between centrosome abnormalities and neurodevelopmental disorders

• Consider both dividing neural progenitors and post-mitotic neurons in experimental design

What are the optimal storage conditions for HAUS3 antibodies?

For maintaining antibody quality:

• Store at -20°C for long-term preservation

• Avoid repeated freeze-thaw cycles by preparing small aliquots

• Store diluted working solutions at 4°C for short-term use only

• Some formulations contain 50% glycerol, which prevents freezing at -20°C

• 0.02% sodium azide is typically included as a preservative

• Typical shelf life is 12 months from receipt when properly stored

What formulation considerations should researchers be aware of?

Antibody formulations typically include:

• PBS buffer at pH 7.3 as the base solution

• 0.02% sodium azide as a preservative

• 50% glycerol for cryoprotection and stability

• Some formulations may include BSA as a carrier protein

• The typical concentration is 0.05-2 mg/ml depending on the supplier

Understanding these formulation details is important when planning downstream applications, particularly those sensitive to buffer components or requiring specific antibody concentrations.

How should unexpected experimental results be documented and reported?

When documenting unexpected findings:

• Maintain detailed records of all experimental conditions

• Document antibody lot numbers, as performance can vary between lots

• Compare results with published literature and known HAUS3 biology

• Consider reaching out to antibody manufacturers for technical support

• Design follow-up experiments to systematically investigate anomalous results

• Share experiences with unexpected binding patterns or molecular weights with the research community