KATNB1 Antibody

What is KATNB1 and what is its function in cellular processes?

KATNB1 (katanin p80 WD40-containing subunit B1) is a regulatory protein encoded by the KATNB1 gene in humans. It functions as the 80 kDa accessory protein (p80 subunit) of the Katanin complex, which is a heterodimer consisting of this regulatory subunit and a 60 kDa ATPase catalytic subunit (KATNA1) . Katanin is a critical microtubule-severing enzyme involved in remodeling microtubule-based structures that influence fundamental cellular processes including cell division, motility, morphogenesis, and signaling . Microtubules, which are polymers of alpha and beta tubulin subunits, form the mitotic spindle during cell division and help organize membranous organelles during interphase . KATNB1, as a regulatory component, modulates the microtubule-severing activity of the catalytic subunits.

What applications can KATNB1 antibodies be used for in research?

KATNB1 antibodies have been validated for multiple experimental applications crucial to cell biology research. The primary applications include:

These applications allow researchers to detect, quantify, and localize KATNB1 protein in various experimental contexts . The antibody has demonstrated reactivity with human, mouse, and rat samples, making it versatile for comparative studies across species .

What is the molecular weight of KATNB1 and how does this affect antibody detection?

The calculated molecular weight of KATNB1 is 72 kDa, though its observed molecular weight typically ranges between 72-80 kDa when detected by Western blot . This variation may result from post-translational modifications or alternative splicing. When selecting antibodies and designing experiments, researchers should anticipate detecting KATNB1 within this molecular weight range and consider using appropriate molecular weight markers. Additionally, when validating a new lot of KATNB1 antibody, confirming that the detected band falls within this expected range is an important quality control step to ensure specificity.

How should KATNB1 antibodies be stored and handled to maintain their efficacy?

For optimal performance, KATNB1 antibodies should be stored at -20°C in their recommended storage buffer, which typically includes PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . Under these conditions, antibodies remain stable for one year after shipment. Aliquoting is generally unnecessary for -20°C storage, minimizing freeze-thaw cycles that could degrade the antibody. Some suppliers provide antibodies in small volumes (20 μl) that contain 0.1% BSA to enhance stability . When working with the antibody, it should be thawed gradually at room temperature or 4°C rather than using heat, which may denature the antibody protein.

What are the optimal conditions for using KATNB1 antibodies in immunofluorescence studies?

For successful immunofluorescence studies with KATNB1 antibodies, the following methodological approach is recommended:

• Fixation: Use 4% paraformaldehyde for 15-20 minutes at room temperature to preserve cellular architecture while maintaining antigen accessibility.

• Permeabilization: Treat with 0.2% Triton X-100 for 5-10 minutes to allow antibody access to intracellular antigens.

• Blocking: Apply 5% normal serum (from the species of the secondary antibody) for 1 hour to reduce non-specific binding.

• Primary antibody incubation: Use KATNB1 antibody at dilutions between 1:50-1:500, optimizing for your specific experimental system . Incubate overnight at 4°C for best results.

• Secondary antibody: Apply fluorophore-conjugated secondary antibody at 1:500-1:1000 dilution for 1-2 hours at room temperature, protecting from light.

• Counterstaining: DAPI nuclear staining can help visualize cellular context.

• Mounting: Use anti-fade mounting medium to preserve fluorescence signal.

This protocol has been validated for detecting KATNB1 in HeLa cells , but should be optimized for each specific cell type or tissue being studied.

How can I validate the specificity of a KATNB1 antibody for my experimental system?

Validating antibody specificity is crucial for ensuring reliable experimental results. For KATNB1 antibodies, a comprehensive validation approach should include:

• Positive controls: Test the antibody in cell lines known to express KATNB1, such as A431, HeLa, or HepG2 cells, which have been validated for KATNB1 detection .

• Knockdown/knockout validation: Perform siRNA knockdown or CRISPR/Cas9 knockout of KATNB1 and confirm decreased or absent signal. Published literature has utilized this approach for validating KATNB1 antibodies in at least 3 studies .

• Molecular weight verification: Confirm that the detected protein band appears at the expected molecular weight (72-80 kDa) .

• Multiple antibody verification: Compare results using antibodies from different sources or that recognize different epitopes of KATNB1.

• Immunoprecipitation followed by mass spectrometry: For definitive validation, immunoprecipitate using the KATNB1 antibody and confirm protein identity via mass spectrometry.

• Immunofluorescence pattern analysis: Verify that the subcellular localization pattern matches expected distribution, particularly at spindle poles during mitosis as described in the literature .

This multi-faceted validation approach ensures that experimental findings with KATNB1 antibodies are reliable and reproducible.

What are the best practices for using KATNB1 antibodies in co-immunoprecipitation experiments?

For successful co-immunoprecipitation (co-IP) of KATNB1 and its interacting partners, the following protocol has proven effective:

• Cell lysis: Lyse cells in a buffer containing 10 mM Tris pH 7.4, 100 mM NaCl, and 0.1% Nonidet P-40, supplemented with protease inhibitors .

• Antibody amount: Use 0.5-4.0 μg of KATNB1 antibody per 1.0-3.0 mg of total protein lysate .

• Bead selection: Anti-species antibody (e.g., anti-rabbit) conjugated magnetic beads provide efficient capture with lower background than agarose beads.

• Incubation conditions: Allow 1-hour incubation at 4°C with gentle rotation to form antibody-antigen complexes.

• Washing steps: Perform at least four washes with buffer containing 10 mM Tris pH 7.4, 100 mM NaCl, and 0.1% Nonidet P-40 to reduce non-specific binding .

• Elution: Elute bound proteins by boiling in 1X Laemmli SDS sample buffer.

• Detection: Analyze 6% of inputs, unbound fractions, and eluates by SDS-PAGE and immunoblotting .

When investigating Katanin complex components, it's particularly important to include microtubule-depolymerizing agents like nocodazole in the experimental setup to ensure that interactions detected are direct protein-protein interactions rather than being mediated by microtubules .

How can I study the interaction between KATNB1 and other Katanin subunits in experimental systems?

Studying the interactions between KATNB1 and other Katanin subunits requires a multi-faceted approach. Based on published methodologies, the following experimental strategy is recommended:

• Co-immunoprecipitation from cell extracts: Express tagged versions of Katanin subunits (e.g., LAP-tagged KATNB1 and HA-tagged KATNA1) in mammalian cells, then perform reciprocal co-IPs to confirm interactions . This approach has successfully demonstrated that KATNB1 associates with KATNA1 and KATNAL1 but not with KATNAL2 .

• In vitro binding assays: For direct interaction studies, use in vitro transcribed and translated S35-labeled Katanin subunits. Combine two different Katanin reactions, immunoprecipitate with anti-tag antibody (e.g., anti-HA) conjugated beads, and detect interactions through radiometric analysis . This method has confirmed that KATNB1 binds directly to KATNA1 and KATNAL1.

• Competition assays: To evaluate binding preferences, perform in vitro binding experiments with increasing concentrations of recombinant B subunits. Research has shown that KATNB1 can compete with KATNBL1 for binding to KATNA1 and KATNAL1, reducing KATNBL1 binding from 100% to approximately 20% in a dose-dependent manner .

• Fluorescence microscopy: Use fluorescently tagged Katanin subunits to visualize co-localization in cells, particularly during different cell cycle stages.

These methods have revealed important insights, including the fact that KATNB1 has a higher affinity for KATNA1 and KATNAL1 than KATNBL1 does , which has significant implications for understanding the regulation of microtubule-severing activity.

What approaches can be used to investigate the role of KATNB1 in microtubule-severing activity?

Investigating KATNB1's role in regulating microtubule-severing activity requires specialized assays that directly measure this function. Based on published research, the following methodological approaches are most effective:

• In vitro microtubule-severing assays with TIRFM: This gold-standard approach involves:

  • Immobilizing rhodamine-labeled microtubules on coverslips

  • Adding recombinant Katanin subunits (A and B) at various ratios

  • Monitoring microtubule severing every 10 seconds for 7 minutes using total internal reflection fluorescence microscopy (TIRFM)

  • Quantifying the rate of microtubule loss as a measure of severing activity

• Concentration-dependent activity analysis: Testing different ratios of catalytic (A) and regulatory (B) subunits has revealed that KATNB1 enhances the microtubule-severing activity of KATNA1 and KATNAL1 , while another B-like subunit (KATNBL1) shows concentration-dependent regulatory effects.

• Live-cell imaging: Expressing fluorescently tagged tubulin along with modulated levels of KATNB1 to observe microtubule dynamics in real-time.

• Cellular phenotype analysis: Examining changes in microtubule organization, mitotic spindle formation, and cell division following KATNB1 knockdown or overexpression.

These approaches have demonstrated that KATNB1 significantly enhances the microtubule-severing activity of Katanin catalytic subunits, providing crucial insights into the regulation of microtubule dynamics in cellular processes .

How does KATNB1 localization change during the cell cycle and what methods best capture these dynamics?

KATNB1 exhibits distinct localization patterns throughout the cell cycle that reflect its functional roles in microtubule regulation. To effectively study these dynamics:

• Immunofluorescence with cell cycle markers: Co-stain cells with KATNB1 antibody (1:50-1:500 dilution) and markers for specific cell cycle phases (e.g., phospho-histone H3 for mitosis) . Research has shown that while the related protein KATNBL1 localizes to spindle poles only during mitosis, KATNB1 shows different localization patterns .

• Live cell imaging with fluorescent fusion proteins: Generate stable cell lines expressing LAP-tagged KATNB1 under inducible promoters, as described in research methodologies . This approach allows real-time visualization of KATNB1 movements during cell cycle progression.

• Cell synchronization techniques: Use methods such as double thymidine block or nocodazole treatment to enrich for specific cell cycle phases, then examine KATNB1 localization at these defined stages.

• Super-resolution microscopy: Techniques such as structured illumination microscopy (SIM) or stochastic optical reconstruction microscopy (STORM) provide enhanced resolution to precisely map KATNB1 localization relative to microtubule structures.

• Biochemical fractionation: Separate nuclear, cytoplasmic, and cytoskeletal fractions from cells at different cell cycle stages to quantitatively assess KATNB1 distribution.

Research findings indicate that KATNB1's subcellular localization is regulated in a cell cycle-dependent manner, which correlates with its function in modulating microtubule dynamics during different cellular processes .

What are common issues encountered when using KATNB1 antibodies and how can they be resolved?

Researchers working with KATNB1 antibodies may encounter several challenges. Here are solutions to common problems:

• Weak or no signal in Western blots:

  • Increase antibody concentration (try 1:1000 before moving to more concentrated dilutions up to 1:500)

  • Extend primary antibody incubation time to overnight at 4°C

  • Verify protein transfer efficiency with Ponceau S staining

  • Ensure adequate protein loading (20-50 μg total protein)

  • Try enhanced chemiluminescence (ECL) detection systems with higher sensitivity

• Multiple bands or high background in Western blots:

  • Increase blocking time and/or concentration (5% milk or BSA for 2 hours)

  • Use more stringent washing conditions (increase TBST concentration to 0.1% Tween-20)

  • Decrease primary antibody concentration (try 1:6000 dilution)

  • Ensure sample is fully denatured with adequate SDS and boiling time

• Weak signal in immunofluorescence:

  • Optimize fixation methods (try 4% PFA for cell lines, but consider methanol for certain applications)

  • Improve antigen retrieval if using tissue sections

  • Increase antibody concentration (start at 1:50 dilution)

  • Extend primary antibody incubation to overnight at 4°C

  • Use signal amplification systems (tyramide signal amplification)

• Non-specific staining in immunofluorescence:

  • Increase blocking time and concentration (5% normal serum for 2 hours)

  • Add 0.1-0.3% Triton X-100 to antibody dilution buffer

  • Reduce primary antibody concentration (try 1:500 dilution)

  • Include additional washing steps

Each experimental system may require specific optimization, and it is recommended that researchers titrate the KATNB1 antibody in their testing system to obtain optimal results .

How can I optimize KATNB1 antibody use for different cell types and tissue samples?

Optimizing KATNB1 antibody protocols for different biological samples requires systematic adaptation:

• Cell line optimization:

  • Begin with validated cell lines (A431, HeLa, HepG2)

  • For new cell lines, start with the manufacturer's recommended protocol

  • Perform antibody titration (1:50 to 1:500 for IF; 1:1000 to 1:6000 for WB)

  • Adjust lysis buffers based on cell type (add specific detergents for difficult-to-lyse cells)

  • Consider cell-specific fixation methods (4% PFA works well for most, but methanol may be better for cytoskeletal proteins in some cell types)

• Tissue sample considerations:

  • For tissue sections, optimize antigen retrieval methods (citrate buffer pH 6.0, EDTA buffer pH 9.0, or enzymatic digestion)

  • Extend primary antibody incubation time to 24-48 hours at 4°C for tissue sections

  • Increase washing time and volume for tissues to reduce background

  • Consider tissue-specific blocking agents (add mouse or human serum when studying mouse or human tissues)

  • Test thinner sections (5-7 μm) if penetration issues occur

• Species cross-reactivity optimization:

  • While KATNB1 antibodies show reactivity with human, mouse, and rat samples , optimization is needed for each species

  • For non-validated species, start with higher antibody concentrations and then titrate down

  • Verify epitope conservation through sequence alignment before attempting cross-species applications

• Sample-dependent controls:

  • Include positive control samples (e.g., human testis tissue which shows KATNB1 expression)

  • Run parallel negative controls (omitting primary antibody)

  • Consider using siRNA knockdown controls in the specific cell type being studied

Remember that antibody performance can be sample-dependent, and validation in each experimental system is crucial for reliable results .

What are the best approaches for multiplexing KATNB1 antibodies with other cell cycle or cytoskeletal markers?

Effective multiplexing of KATNB1 with other markers requires careful planning to avoid cross-reactivity and signal interference:

• Antibody selection for multiplexing:

  • Choose primary antibodies raised in different host species (e.g., rabbit anti-KATNB1 with mouse anti-tubulin)

  • Verify that secondary antibodies do not cross-react with non-target primaries

  • Consider directly conjugated primary antibodies for multi-color imaging

  • Test antibodies individually before combining to establish baseline signals

• Optimized staining protocol:

  • Sequential staining may be necessary if antibodies require different fixation methods

  • Begin with the weakest signal antibody first

  • Include additional blocking steps between primary antibody incubations

  • Extend washing steps to minimize non-specific binding

• Recommended marker combinations:

  • KATNB1 + α-tubulin: Reveals relationship between KATNB1 and microtubule structures

  • KATNB1 + cell cycle markers (Ki67, phospho-histone H3): Shows cell cycle-dependent localization

  • KATNB1 + KATNA1: Demonstrates co-localization of Katanin subunits

  • KATNB1 + centrosomal markers (γ-tubulin, pericentrin): Highlights spindle pole association

• Imaging considerations:

  • Use sequential scanning for confocal microscopy to prevent bleed-through

  • Establish proper exposure settings using single-stained controls

  • Consider spectral unmixing for fluorophores with overlapping spectra

  • Use appropriate filter sets to clearly separate fluorescent signals

This multiplexing approach has been successfully applied in studies examining the relationship between KATNB1 and microtubule dynamics during cell division , providing valuable insights into the spatial and temporal regulation of microtubule-severing activity.

How do post-translational modifications affect KATNB1 function and antibody detection?

Post-translational modifications (PTMs) of KATNB1 represent an important regulatory mechanism that can influence both its function and detection by antibodies:

• Impact on antibody detection:

  • The observed molecular weight range of KATNB1 (72-80 kDa) suggests the presence of PTMs that alter protein migration on SDS-PAGE

  • Phosphorylation, a common regulatory modification of cell cycle proteins, can cause band shifts in Western blots

  • Epitopes containing modified residues may show reduced antibody binding, potentially leading to false-negative results

  • Researchers should consider using phosphatase treatment of samples when inconsistent results occur

• Functional significance:

  • Phosphorylation of KATNB1 may regulate its binding affinity to KATNA1 and KATNAL1

  • Cell cycle-dependent modifications likely control KATNB1's ability to enhance or inhibit microtubule-severing activity

  • PTMs may influence KATNB1's subcellular localization, particularly during mitosis

  • Competition between KATNB1 and KATNBL1 for binding to catalytic subunits could be regulated through differential modification

• Methodological approaches to study PTMs:

  • Phospho-specific antibodies can identify specific modified residues

  • Mass spectrometry following immunoprecipitation can comprehensively map PTMs

  • Mutagenesis of candidate modification sites can confirm functional significance

  • In vitro kinase assays can identify enzymes responsible for KATNB1 modification

Understanding the PTM landscape of KATNB1 will provide critical insights into the temporal and spatial regulation of microtubule-severing activity throughout the cell cycle and in different cellular contexts.

What are the implications of the competitive binding between KATNB1 and KATNBL1 for microtubule regulation?

The competitive binding between KATNB1 and KATNBL1 to catalytic Katanin subunits represents a sophisticated regulatory mechanism for microtubule dynamics:

• Molecular basis of competition:

  • Both KATNB1 and KATNBL1 directly bind to KATNA1 and KATNAL1, but not to KATNAL2

  • KATNB1 demonstrates higher binding affinity, capable of reducing KATNBL1 binding from 100% to approximately 20% in dose-dependent competition assays

  • Conversely, KATNBL1 only weakly competes against KATNB1, reducing its binding from 100% to approximately 84%

  • No direct interaction occurs between KATNB1 and KATNBL1

• Functional consequences:

  • KATNB1 enhances the microtubule-severing activity of catalytic subunits

  • KATNBL1 demonstrates concentration-dependent effects: at low concentrations (1:0.03125-1:0.0625 ratio with KATNAL1) it enhances severing activity, while at higher concentrations (1:0.125-1:0.5) it inhibits activity

  • This competitive system allows for fine-tuning of microtubule-severing activity based on the relative abundance of regulatory subunits

• Cell cycle implications:

  • KATNBL1 localizes to spindle poles specifically during mitosis, while showing nuclear sequestration during interphase through an N-terminal nuclear localization signal

  • This suggests that the competition between KATNB1 and KATNBL1 may vary throughout the cell cycle

  • The temporal regulation of this competition likely coordinates microtubule remodeling during specific cellular processes

This competitive binding mechanism represents a previously unappreciated layer of regulation for Katanin activity, with significant implications for understanding microtubule dynamics in both normal cellular processes and pathological conditions .

How can CRISPR/Cas9 genome editing be combined with KATNB1 antibodies to study protein function?

CRISPR/Cas9 genome editing technology offers powerful approaches for studying KATNB1 function when combined with antibody-based detection methods:

• Generation of endogenously tagged KATNB1:

  • Design CRISPR/Cas9 system to introduce fluorescent tags (GFP, mCherry) or epitope tags (FLAG, HA) at the endogenous KATNB1 locus

  • This preserves physiological expression levels and regulatory elements

  • Combined with validated antibodies, this approach allows correlation between endogenous protein behavior and antibody detection

  • Live-cell imaging of tagged protein can be complemented with fixed-cell antibody staining for multiplexing with other markers

• Domain-specific mutants and truncations:

  • Create precise mutations in functional domains (e.g., WD40 repeat regions) of endogenous KATNB1

  • Antibody detection can then assess how these mutations affect localization, interaction with KATNA1/KATNAL1, and competition with KATNBL1

  • The LAP-tagging approach used in published studies can be adapted for CRISPR/Cas9-mediated genome editing

• Validation and control strategies:

  • Generate complete KATNB1 knockout cell lines as negative controls for antibody specificity

  • Create isogenic cell lines with varying mutations to compare antibody reactivity

  • Perform rescue experiments with wild-type or mutant KATNB1 to confirm phenotype specificity

• Advanced experimental applications:

  • Auxin-inducible degron (AID) tagging of KATNB1 allows temporal control of protein depletion

  • APEX2 proximity labeling combined with KATNB1 antibody validation enables mapping of protein interaction landscapes

  • CRISPR interference (CRISPRi) or activation (CRISPRa) can modulate KATNB1 expression levels without protein modification

These approaches, combined with rigorous antibody validation, provide unprecedented opportunities to dissect the functions of KATNB1 in microtubule regulation and broader cellular processes.

How do different commercial KATNB1 antibodies compare in terms of specificity and application range?

When selecting a KATNB1 antibody for research, understanding the differences between available options is critical:

• Host species and antibody type:

  • Rabbit polyclonal antibodies (e.g., 14969-1-AP from Proteintech, HPA041165 from Atlas Antibodies) offer high sensitivity but potential batch-to-batch variation

  • Mouse monoclonal antibodies (e.g., clone 3B6 from antibodies-online) provide consistent specificity but potentially lower epitope coverage

  • Each antibody type has advantages for different applications, with polyclonals often preferred for detection and monoclonals for specific epitope targeting

• Validated applications comparison:

• Immunogen and epitope considerations:

  • Different antibodies target distinct regions of the KATNB1 protein

  • Proteintech's antibody uses KATNB1 fusion protein Ag6836 as the immunogen

  • Epitope differences may affect detection of specific KATNB1 isoforms or modified forms

  • For studying protein interactions, epitope location relative to binding domains is an important consideration

• Specificity validation level:

  • Published literature citations (8 for WB, 7 for IF with Proteintech antibody)

  • Knockout/knockdown validation (3 publications for Proteintech antibody)

  • Tissue expression pattern validation (testis and pancreas for Atlas Antibodies)

When designing experiments, researchers should select antibodies based on the specific application requirements and validation level appropriate for their research questions.

What are the advantages and limitations of antibody-based versus genetic tagging approaches for studying KATNB1?

Both antibody-based detection and genetic tagging approaches offer distinct advantages for studying KATNB1:

• Antibody-based detection:

  • Advantages:

    • Detects endogenous protein at physiological levels

    • No genetic manipulation required

    • Compatible with primary tissues and clinical samples

    • Multiple epitopes can be targeted with different antibodies

  • Limitations:

    • Potential for cross-reactivity or non-specific binding

    • Fixation requirements may alter protein conformation

    • Limited to fixed samples for immunofluorescence

    • Batch-to-batch variation (particularly with polyclonal antibodies)

• Genetic tagging approaches:

  • Advantages:

    • Live-cell imaging capability

    • High specificity due to direct fusion to protein of interest

    • Can track protein dynamics in real time

    • The LAP-tagging system used for KATNB1 in published research allows for consistent expression from a defined genomic locus

  • Limitations:

    • Tag may interfere with protein function

    • Expression levels may differ from endogenous protein

    • Limited to genetically manipulable systems

    • Time-consuming to generate stable cell lines

• Complementary approaches:

  • Validation of antibody specificity using tagged protein as a reference

  • Using antibodies to detect endogenous protein in conjunction with live imaging of tagged protein

  • Confirming antibody-based observations with genetic approaches and vice versa

  • Employing CRISPR/Cas9 knock-in strategies to tag endogenous KATNB1 while maintaining physiological regulation

The optimal approach depends on the specific research question, with many studies benefiting from combining both methodologies to leverage their complementary strengths.

What is the potential role of KATNB1 in neurodevelopmental disorders and how can antibodies help investigate this?

Recent research has implicated KATNB1 in neurodevelopmental processes and associated disorders:

• Neurological significance of KATNB1:

  • Microtubule severing plays critical roles in neurite formation, neuronal migration, and axon guidance

  • Mutations in microtubule-severing enzymes have been linked to neurodevelopmental disorders

  • KATNB1's regulatory function in the Katanin complex suggests its involvement in the precise control of neuronal microtubule dynamics

• Antibody-based investigative approaches:

  • Immunohistochemistry with KATNB1 antibodies in normal versus diseased brain tissue sections

  • Tracking KATNB1 expression patterns during neuronal differentiation and migration using IF/ICC (1:50-1:500 dilution)

  • Co-localization studies with neuronal markers to map KATNB1 distribution in different neural cell types

  • Examining KATNB1 expression in induced pluripotent stem cell (iPSC)-derived neurons from patients with neurodevelopmental disorders

• Functional studies combining antibodies and other techniques:

  • Using validated KATNB1 antibodies to assess protein levels following introduction of patient-derived mutations

  • Combining CRISPR/Cas9 genome editing of KATNB1 with antibody-based detection to study functional consequences

  • Live imaging of neuronal cultures with fluorescently tagged KATNB1 followed by fixation and antibody staining for other markers

  • Investigating the interaction between KATNB1 and other neuronal proteins using co-IP with KATNB1 antibodies (0.5-4.0 μg per IP)

Understanding KATNB1's role in neurodevelopment could provide insights into the pathogenesis of developmental disorders and potentially identify new therapeutic targets.

How might proteomics approaches combined with KATNB1 antibodies advance understanding of the "Katan-ome"?

The integration of proteomics with KATNB1 antibodies offers powerful approaches to comprehensively map the "Katan-ome" (Katanin family interaction network):

• Immunoprecipitation-mass spectrometry (IP-MS):

  • Using validated KATNB1 antibodies (0.5-4.0 μg) to immunoprecipitate native protein complexes

  • Coupling this with high-resolution mass spectrometry to identify all interacting proteins

  • Comparing interactomes under different cellular conditions (e.g., cell cycle stages, differentiation states)

  • Cross-referencing with published proteomic analyses of Katanin family members

• Proximity labeling proteomics:

  • Expressing KATNB1 fused to proximity labeling enzymes (BioID, APEX2)

  • Using antibodies to validate expression and localization of fusion proteins

  • Identifying proteins in close proximity to KATNB1 in living cells

  • Comparing results with traditional IP-MS to distinguish stable versus transient interactions

• Dynamic interaction analysis:

  • Combining SILAC (Stable Isotope Labeling with Amino acids in Cell culture) with KATNB1 antibody IP

  • Quantifying changes in protein-protein interactions under different conditions

  • Investigating how the competition between KATNB1 and KATNBL1 for binding to catalytic subunits is regulated

  • Identifying post-translational modifications that influence interaction dynamics

• Validation and functional characterization:

  • Using antibodies against newly identified interaction partners for reciprocal co-IP

  • Performing co-localization studies with KATNB1 and novel interactors

  • Investigating how disruption of specific interactions affects microtubule-severing activity

This integrated approach would significantly expand our understanding of the complex regulatory network controlling microtubule severing in diverse cellular contexts.

What databases and bioinformatic resources are available for KATNB1 research planning?

Researchers investigating KATNB1 can leverage various bioinformatic resources to inform experimental design:

• Protein information databases:

  • UniProt (ID: Q9BVA0) provides comprehensive protein information including sequence, domains, and post-translational modifications

  • NCBI Gene (ID: 10300) offers genomic context, expression data, and literature references

  • Human Protein Atlas presents tissue expression patterns and subcellular localization data

• Antibody validation resources:

  • Antibodypedia aggregates validation data for commercial antibodies

  • CiteAb provides citation metrics for antibodies in published literature

  • Proteintech, Atlas Antibodies, and other manufacturers' validation galleries show application-specific results

• Protein interaction databases:

  • BioGRID, STRING, and IntAct databases catalog known protein-protein interactions

  • The published "Katan-ome" network from proteomic studies provides a foundation for investigation

• Expression databases:

  • GTEx Portal shows tissue-specific expression patterns

  • Cancer Cell Line Encyclopedia (CCLE) provides expression data across cancer cell lines

  • Single-cell RNA sequencing databases reveal cell type-specific expression

• Structural resources:

  • PDB (Protein Data Bank) for structural information, when available

  • AlphaFold Protein Structure Database for predicted structures

  • ModBase for homology models of protein domains

These resources help researchers make informed decisions about experimental design, including cell line selection, antibody choice, and interaction partners to investigate when studying KATNB1.

What are recommended experimental controls when working with KATNB1 antibodies?

Proper experimental controls are essential for generating reliable data with KATNB1 antibodies:

• Positive controls:

  • Cell lines with confirmed KATNB1 expression (A431, HeLa, HepG2 cells)

  • Tissue samples with known expression (testis, pancreas)

  • Recombinant KATNB1 protein as a Western blot standard

  • GFP-tagged KATNB1 expression as a parallel verification system

• Negative controls:

  • KATNB1 knockout or knockdown samples (siRNA, CRISPR/Cas9)

  • Primary antibody omission in immunostaining

  • Isotype control antibodies (rabbit IgG for polyclonal antibodies)

  • Cell lines with low or no KATNB1 expression

• Specificity controls:

  • Peptide competition/blocking with immunizing antigen

  • Multiple antibodies targeting different epitopes

  • Western blot band at expected molecular weight (72-80 kDa)

  • Comparison of staining pattern with published localization data

• Application-specific controls:

  • For immunoprecipitation: 6% input and unbound fractions

  • For immunofluorescence: co-staining with established markers

  • For Western blot: loading controls (β-actin, GAPDH)

  • For microtubule-severing assays: buffer-only and catalytic subunit-only conditions

• Validation controls:

  • Correlation between protein and mRNA levels

  • Concordance between different detection methods

  • Reproducibility across multiple experimental replicates