HAUS4 Antibody

What is HAUS4 and why is it significant in cell biology research?

HAUS4 (also known as C14orf94) is a subunit of the centrosome complex termed the human augmin complex. This protein localizes to spindle microtubules and plays crucial roles in mitotic spindle assembly, maintenance of centrosome integrity during cell division, and completion of cytokinesis . HAUS4 is part of an 8-subunit complex (HAUS1-8) that functions in microtubule-dependent amplification of centrosomes . Studying HAUS4 is significant for understanding fundamental cellular processes related to mitosis and chromosomal stability, which have implications in cancer research and cell biology.

What are the optimal conditions for Western blot detection of HAUS4?

For optimal Western blot detection of HAUS4:

• Sample preparation: Use tissues with known HAUS4 expression (brain, placenta, lung, skin)

• Protein loading: 20-30 μg of total protein is typically sufficient

• Antibody dilution: Use 1:1000-1:4000 dilution for primary antibody

• Molecular weight expectation: Be prepared to detect bands at multiple molecular weights (28 kDa, 37 kDa, 42 kDa) due to different isoforms

• Blocking: 5% non-fat milk in TBST for 1 hour at room temperature

• Incubation: Overnight at 4°C for primary antibody followed by appropriate secondary antibody

• Detection method: Enhanced chemiluminescence (ECL) is suitable

For troubleshooting, note that small volumes of antibody may occasionally become entrapped in the seal of the product vial during shipment and storage. If necessary, briefly centrifuge the vial to dislodge any liquid in the container's cap .

How should I optimize immunohistochemistry protocols for HAUS4 detection in different tissue samples?

For optimal immunohistochemistry detection of HAUS4:

• Antigen retrieval: Use TE buffer pH 9.0 (alternatively, citrate buffer pH 6.0)

• Antibody dilution: Start with 1:100-1:500

• Tissue selection: HAUS4 antibodies have been validated on human prostate cancer tissue, human colon cancer tissue, and human placenta tissue

• Incubation conditions: Typically overnight at 4°C

• Detection system: Use a polymer-based detection system for enhanced sensitivity

• Controls: Include positive tissues (placenta, skin) and negative controls (antibody diluent only)

• Signal amplification: Consider tyramide signal amplification for low-expressing samples

Each new tissue type may require optimization of antigen retrieval conditions and antibody concentration.

How can I verify the specificity of my HAUS4 antibody and troubleshoot non-specific binding issues?

To verify specificity and address non-specific binding:

• Peptide competition assay: Pre-incubate the antibody with the immunogen peptide before application to block specific binding

• HAUS4 knockdown/knockout controls: Use CRISPR-edited cells with HAUS4 knockout as negative controls

• Multiple antibody validation: Test different HAUS4 antibodies targeting different epitopes

• Cross-reactivity testing: Check binding in samples known to lack HAUS4 expression

• Blocking optimization: Test different blocking agents (BSA, normal serum, commercial blockers)

• Antibody titration: Perform a dilution series to find the optimal concentration that maximizes specific signal while minimizing background

• Secondary antibody controls: Include controls with only secondary antibody to identify non-specific binding

For reproducibility, always document lot numbers and validation data of antibodies used.

What are common pitfalls in HAUS4 antibody-based experiments and how can they be avoided?

Common pitfalls and their solutions:

• Molecular weight discrepancies:

  • Pitfall: HAUS4 appears at different molecular weights (28, 37, 42 kDa)

  • Solution: Check all potential molecular weights and verify with positive controls

• Subcellular localization variability:

  • Pitfall: HAUS4 localization changes during cell cycle (centrosome, spindle microtubules)

  • Solution: Synchronize cells or co-stain with cell cycle markers

• Fixation sensitivity:

  • Pitfall: Some epitopes may be sensitive to particular fixatives

  • Solution: Compare different fixation methods (PFA, methanol, acetone)

• Antibody specificity issues:

  • Pitfall: Cross-reactivity with other HAUS family members

  • Solution: Validate with gene silencing or knockout approaches

• Storage and handling:

  • Pitfall: Repeated freeze-thaw cycles can degrade antibody quality

  • Solution: Aliquot antibodies and store at -20°C or -80°C

How can I design co-immunoprecipitation experiments to study HAUS4 protein interactions with other components of the augmin complex?

To study HAUS4 interactions within the augmin complex:

• Lysis buffer optimization:

  • Use gentle lysis buffers (e.g., 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% Triton X-100)

  • Include protease and phosphatase inhibitors

  • For microtubule-associated proteins, consider adding stabilizing agents like taxol

• Antibody selection:

  • Choose antibodies with demonstrated IP capability

  • Consider epitope-tagged HAUS4 constructs if antibody IP efficiency is low

• Co-IP protocol:

  • Pre-clear lysates with protein A/G beads

  • Incubate with HAUS4 antibody overnight at 4°C

  • Capture with protein A/G beads for 2-3 hours

  • Wash extensively (at least 4-5 times)

  • Elute using either low pH, high pH, or SDS sample buffer

• Controls:

  • Include IgG control

  • Include input fraction (5-10% of starting material)

  • Consider reciprocal IP with antibodies against known HAUS complex members

• Analysis:

  • Western blot for other HAUS subunits (HAUS1-8)

  • Consider mass spectrometry for unbiased interactome analysis

What advanced imaging techniques are most effective for studying HAUS4 localization and dynamics during mitosis?

Advanced imaging techniques for HAUS4 dynamics:

• Super-resolution microscopy (SRM):

  • Structured Illumination Microscopy (SIM): Provides 2x resolution improvement

  • Stimulated Emission Depletion (STED): For visualizing fine HAUS4 distribution at centrosomes

  • Photoactivated Localization Microscopy (PALM)/Stochastic Optical Reconstruction Microscopy (STORM): For single-molecule level resolution

• Live-cell imaging:

  • Generate fluorescent protein-tagged HAUS4 constructs (e.g., GFP-HAUS4)

  • Use spinning disk confocal microscopy for reduced phototoxicity

  • Consider lattice light-sheet microscopy for highest temporal resolution

• Multi-color imaging:

  • Co-label with markers for:

    • Centrosomes (γ-tubulin, pericentrin)

    • Microtubules (α-tubulin)

    • Chromosomes (DAPI, H2B-GFP)

    • Cell cycle markers (PCNA, cyclin B)

• Fluorescence Recovery After Photobleaching (FRAP):

  • Measure HAUS4 dynamics at the mitotic spindle

  • Determine exchange rates and stable vs. mobile fractions

• Expansion microscopy:

  • Physical expansion of samples can provide increased resolution with standard confocal microscopy

  • Particularly useful for crowded structures like centrosomes

How do I correctly interpret HAUS4 antibody staining patterns in the context of cell cycle progression?

To correctly interpret HAUS4 staining patterns:

• Cell cycle-dependent localization:

  • Interphase: HAUS4 localizes to interphase centrosomes

  • Mitosis: HAUS4 localizes to spindle microtubules

• Co-localization analysis:

  • With centrosome markers (γ-tubulin, pericentrin): Should show partial overlap

  • With microtubule markers (α-tubulin): Should show strong co-localization along spindle fibers

  • With kinetochore markers: May show proximity but not direct co-localization

• Quantitative assessment:

  • Measure HAUS4 signal intensity at centrosomes across cell cycle stages

  • Assess spindle-associated HAUS4 vs. cytoplasmic HAUS4 ratio

• Abnormal patterns:

  • Diffuse cytoplasmic staining: May indicate inactive protein or antibody issues

  • Asymmetric centrosomal staining: Could indicate centrosome abnormalities

  • Nuclear staining: Likely non-specific unless validated

• Distinguishing isoforms:

  • Different isoforms (27-29 kDa, 37 kDa, 42 kDa) may show slightly different localization patterns

  • Use isoform-specific antibodies when available

What approaches can be used to study the functional relationship between HAUS4 and other components of the centrosome and mitotic apparatus?

To study functional relationships between HAUS4 and other centrosomal components:

• Genetic perturbation approaches:

  • CRISPR/Cas9-mediated knockout

  • siRNA or shRNA-mediated knockdown

  • Overexpression of dominant-negative mutants

  • Generation of separation-of-function mutants

• Interaction mapping:

  • Proximity ligation assay (PLA) to detect protein-protein interactions in situ

  • BioID or TurboID proximity labeling to identify proximity interactions

  • FRET/FLIM for direct interaction detection in live cells

• Functional readouts:

  • Spindle morphology (bipolar vs. multipolar spindles)

  • Microtubule nucleation assays

  • Centrosome integrity (measured by centrosome marker intensity)

  • Mitotic timing and progression (measured by live-cell imaging)

  • Chromosome segregation fidelity (measured by aneuploidy rates)

• Biochemical reconstitution:

  • In vitro reconstitution of partial or complete HAUS complex

  • Microtubule binding and bundling assays

  • Microtubule nucleation assays with purified components

• Electron microscopy:

  • Immune-electron microscopy to determine precise localization

  • Correlative light and electron microscopy (CLEM) to combine functional data with ultrastructural information

How do different fixation and permeabilization methods affect HAUS4 antibody staining efficiency in immunofluorescence?

Different fixation methods impact HAUS4 antibody staining:

Always perform side-by-side comparisons with multiple fixation methods when establishing protocols for a new cell type.

What strategies can be employed when HAUS4 antibody experimental results conflict with published data?

When facing conflicting HAUS4 antibody results:

• Antibody validation reassessment:

  • Perform peptide competition assays

  • Test in HAUS4 knockout/knockdown cells

  • Compare multiple antibodies targeting different epitopes

  • Validate using orthogonal methods (e.g., tagged HAUS4 expression)

• Technical parameters examination:

  • Cell synchronization status (HAUS4 localization is cell cycle-dependent)

  • Fixation and permeabilization conditions

  • Detection system sensitivity

  • Species differences (human vs. mouse vs. rat reactivity)

• Biological variation sources:

  • Cell line differences (transformed vs. primary cells)

  • Tissue-specific isoform expression

  • Cellular stress conditions

  • Cell density and culture conditions

• Data integration approaches:

  • Perform meta-analysis of published results

  • Contact authors of conflicting papers for protocols

  • Combine multiple detection methods (IF, IP-Western, live imaging)

  • Quantitative analysis rather than qualitative assessment

• Reporting conflicts transparently:

  • Document all experimental conditions

  • Present both your data and the conflicting published data

  • Propose biological or technical explanations for discrepancies

  • Consider cell/tissue-specific regulation may explain differences

By systematically evaluating these factors, you can determine whether conflicts arise from technical issues or represent genuine biological differences worth further investigation.