CLASP1 Antibody

What is CLASP1 and what cellular functions does it regulate?

CLASP1 is a microtubule plus-end tracking protein (+TIP) that plays crucial roles in regulating microtubule dynamics and organization. It contains a serine/arginine-rich domain with two short SxIP motifs that facilitate interaction with EB-proteins and microtubule ends . CLASP1 is involved in multiple cellular processes including spindle formation during cell division, cell migration, and intracellular transport. In neurons, it has been implicated in neurite outgrowth and axon extension . CLASP1 localizes to various cellular compartments including chromosomes, cytoplasm, Golgi apparatus, centromeres, centrosomes, kinetochores, microtubule organizing centers, spindles, and the trans-Golgi network . Understanding these localizations is essential for properly interpreting immunostaining results in your experiments.

What applications are CLASP1 antibodies typically used for in research?

CLASP1 antibodies are versatile research tools validated for multiple applications. According to available product information, they are commonly used for Western blotting (WB), immunohistochemistry (IHC), immunocytochemistry (ICC), immunofluorescence (IF), and enzyme-linked immunosorbent assay (ELISA) . When selecting a CLASP1 antibody, researchers should verify the specific applications for which it has been validated. For example, the Boster Bio Anti-CLASP1 Antibody (A30619) has been tested in WB, IHC, ICC, IF, and ELISA applications with recommended dilutions of 1:500-1:2000 for WB, 1:100-1:300 for IHC, and 1:40000 for ELISA . These application-specific dilutions are critical starting points for optimizing your experimental protocols.

What species reactivity should I consider when selecting a CLASP1 antibody?

When selecting a CLASP1 antibody, carefully consider the species of your experimental model. Based on available products, many CLASP1 antibodies show reactivity against human and mouse samples . Some antibodies, like the Assay Genie CLASP1 Rabbit Polyclonal Antibody (CAB7081), also react with rat samples . It's important to note that even if an antibody has not been explicitly validated for your species of interest, there may be sufficient homology for cross-reactivity. For example, a researcher inquired whether an anti-CLASP1 antibody (A30619) validated for human and mouse would work in horse tissues, and while not specifically validated, there was a possibility of cross-reactivity due to protein conservation across species . When working with unconventional model organisms, consider testing multiple antibodies or contacting manufacturers for cross-reactivity information.

What is the difference between CLASP1 and CLASP2, and how does this affect antibody selection?

CLASP1 and CLASP2 are homologous +TIP proteins encoded by different genes . They share structural similarities including a serine/arginine-rich domain with SxIP motifs for EB-protein interaction and a C-terminal protein-protein interaction domain that binds partners like CLIP-115 and CLIP-170 . Due to these similarities, antibodies raised against one protein may cross-react with the other to some extent. For example, antibodies #402 and #2292 raised against CLASP1 show some cross-reactivity with GFP-CLASP2α, while antibody #2358 raised against CLASP2 cross-reacts somewhat with GFP-CLASP1α .

When selecting antibodies for experiments requiring specific detection of either CLASP1 or CLASP2, it's crucial to review the cross-reactivity data and possibly perform control experiments with overexpression constructs. For studies investigating the distinct functions of these proteins, consider using siRNA approaches targeting each protein separately, as demonstrated in previous research .

How can I verify CLASP1 antibody specificity in my experimental system?

Verifying antibody specificity is crucial for reliable research outcomes. Several approaches can be implemented:

• Peptide blocking controls: As demonstrated with the Boster antibody, specificity can be validated by blocking with the synthesized peptide used as the immunogen. Images from western blot and immunohistochemistry analyses show signal elimination when the antibody is pre-incubated with the blocking peptide .

• siRNA knockdown validation: Using CLASP1-specific siRNAs to reduce endogenous protein levels can confirm antibody specificity. Previous research has employed multiple siRNAs (CLASP1#A and CLASP1#B) to achieve approximately 70% reduction in CLASP1 levels, creating a hypomorphic state that can be detected by Western blotting and immunofluorescence . Following siRNA treatment, antibody signals should be significantly reduced compared to control transfections.

• FACS analysis: Flow cytometry can be used to assess the homogeneity of antibody staining after CLASP1 knockdown, as previously demonstrated with CLASP1+2 knockdown cells .

• Cross-reactivity testing: If studying both CLASP1 and CLASP2, test the antibody against overexpressed GFP-tagged versions of both proteins to quantify potential cross-reactivity .

These validation approaches are essential when working with new cell lines or tissues, as they ensure that observed signals genuinely represent CLASP1 rather than non-specific interactions or related proteins.

What strategies can resolve discrepancies between observed molecular weight and predicted size of CLASP1?

Researchers often encounter differences between observed and predicted molecular weights of CLASP1. According to product information, while the calculated molecular weight of CLASP1 is approximately 169 kDa, western blots may detect bands at different sizes, such as 72 kDa . These discrepancies can arise from several factors:

• Protein isoforms: CLASP1 exists in multiple isoforms, including CLASP1α, which appears at approximately 160 kDa in HeLa cells . Verify which isoform your antibody targets by checking the immunogen sequence information.

• Post-translational modifications: Phosphorylation and other modifications can alter protein migration. CLASP1 is regulated by phosphorylation, which can affect its apparent molecular weight.

• Proteolytic processing: Some antibodies may detect cleaved forms of CLASP1, depending on the epitope location and sample preparation methods.

• Antibody specificity issues: Validate bands using knockdown approaches or peptide competition assays to confirm specificity.

When reporting results, clearly indicate the observed molecular weight alongside the predicted size, and provide potential explanations for any discrepancies. If possible, include controls with overexpressed full-length CLASP1 to establish the migration pattern in your gel system.

How can I optimize CLASP1 antibody protocols for challenging tissues or cell types?

Optimizing CLASP1 antibody protocols for challenging samples requires systematic approach:

• Fixation optimization: For immunofluorescence or immunohistochemistry, test different fixatives (paraformaldehyde, methanol, or combinations) as CLASP1 epitopes may be sensitive to specific fixation methods. Microtubule-associated proteins often require specific fixation protocols to preserve native structure.

• Antigen retrieval methods: For paraffin-embedded tissues, compare heat-induced epitope retrieval (HIER) with different buffers (citrate, EDTA, Tris) to unmask CLASP1 epitopes.

• Blocking optimization: Increase blocking agent concentration (5-10% BSA or serum) and duration (2-3 hours) to reduce background in high-autofluorescence tissues.

• Signal amplification: For low-abundance detection, consider tyramide signal amplification (TSA) or polymer-based detection systems.

• Antibody concentration titration: Perform systematic dilution series beyond manufacturer recommendations. For example, while Boster recommends 1:100-1:300 for IHC, challenging tissues might require 1:50 or even 1:500 depending on expression levels .

• Incubation conditions: Test both overnight incubation at 4°C and room temperature incubations of varying durations.

Document all optimization steps methodically, as successful parameters may vary significantly between tissue types and experimental conditions.

What are the optimal conditions for using CLASP1 antibodies in immunofluorescence studies?

For optimal immunofluorescence results with CLASP1 antibodies, consider these methodological aspects:

For specialized applications like visualizing CLASP1 during mitosis, additional steps such as synchronizing cells or performing specific fixation protocols optimized for preserving mitotic structures may be necessary.

What are the key considerations for Western blot analysis of CLASP1?

Successful Western blot analysis of CLASP1 requires attention to several technical details:

• Sample preparation: Due to CLASP1's large size and association with cytoskeletal elements, use strong lysis buffers containing SDS and mechanical disruption to ensure complete extraction. Include phosphatase inhibitors if studying CLASP1 phosphorylation status.

• Gel selection: Use low percentage (6-8%) gels or gradient gels to properly resolve the full-length CLASP1 (approximately 169 kDa).

• Transfer conditions: For large proteins like CLASP1, extend transfer times or use specialized protocols for high molecular weight proteins (overnight transfer at low voltage or semi-dry transfer systems optimized for large proteins).

• Antibody dilution: Begin with the manufacturer's recommended dilution range (1:500-1:2000 for WB with the Boster antibody) and optimize based on your specific samples. Lower dilutions (1:500) may be necessary for detecting endogenous CLASP1 in cell types with lower expression.

• Positive controls: Include lysates from cells known to express CLASP1, such as SH-SY5Y, Jurkat, HepG2, or MCF7 cells .

• Expected bands: Be prepared to observe bands at different molecular weights. While the calculated molecular weight is approximately 169 kDa, observed bands may appear at different sizes (e.g., 72 kDa has been reported) . Document all bands and validate specificity with appropriate controls.

• Blocking conditions: Optimize blocking conditions (5% milk or BSA) and duration to minimize background while preserving specific signals.

For detecting specific phosphorylated forms of CLASP1, consider using phospho-specific antibodies if available, or phosphatase treatments as controls.

How do CLASP1 and CLASP2 cooperate or function distinctly in neuronal development?

Research on CLASP1 and CLASP2 in neuronal development reveals complex, sometimes contradictory roles that researchers should consider when designing experiments:

• Species-specific functions: In Xenopus, depletion of XCLASP1 (which resembles human CLASP1) impedes axon extension . This contrasts with mammalian models, where CLASP knockdown effects vary by neuronal type and specific paralog.

• Neuronal type-specific functions: In mammalian neurons, CLASP paralog functions differ based on the specific neuronal population:

  • In cortical neurons: CLASP2 knockdown results in longer axons

  • In hippocampal neurons (HNs): CLASP2 knockdown leads to shorter axons

  • In dorsal root ganglia (DRG) neurons: CLASP1 knockdown or combined CLASP1/2 depletion impedes neurite outgrowth, while single CLASP2 knockdown has less pronounced effects

• Molecular interactions: When studying CLASP1 in neurons, consider its interactions with neuron-specific partners. CLASP1 interacts with the adaptor protein Dab1 , which is involved in Reelin signaling during neuronal migration and positioning.

These complex, context-dependent functions highlight the importance of carefully selecting appropriate neuronal models and considering both CLASP1 and CLASP2 when studying neuronal development. Researchers should design experiments that can distinguish between their unique and redundant functions, potentially using individual and combined knockdown approaches.

What is known about CLASP1's role in mitosis and how can antibodies help investigate this function?

CLASP1 plays critical roles during mitosis that can be investigated using appropriate antibody-based approaches:

• Localization during mitosis: CLASP1 dynamically localizes to key mitotic structures. Immunofluorescence studies show CLASP1 at kinetochores, spindle microtubules, and centrosomes during different mitotic phases . When investigating these localizations, optimize fixation protocols to preserve mitotic structures.

• Functional redundancy with CLASP2: Studies indicate that CLASP1 and CLASP2 cooperate during mitosis. Both proteins are coexpressed in proliferating cells, as confirmed by RT-PCR analysis in various tumor-derived cell lines . When designing experiments, consider this redundancy and potential compensatory mechanisms.

• High-resolution imaging approaches: For detailed analysis of CLASP1 localization during mitosis, consider specialized imaging techniques. Immuno-electron microscopy has been used to visualize EGFP-CLASP1 in mitotic cells, using anti-GFP antibodies and gold-conjugated secondary antibodies . This approach provides nanometer-scale resolution of CLASP1 positioning relative to mitotic structures.

• Knockdown studies: Combined knockdown of CLASP1 and CLASP2 can be used to investigate their roles in mitotic progression. Previous studies achieved approximately 70% reduction of both proteins using specific siRNAs , creating a hypomorphic condition suitable for studying mitotic defects.

When designing experiments to study CLASP1 in mitosis, use proper synchronization methods to enrich for mitotic cells, and consider live-cell imaging with fluorescently tagged CLASP1 to complement fixed-cell antibody-based approaches.

What are the key specifications and applications of commercially available CLASP1 antibodies?

Below is a comparative table of commercially available CLASP1 antibodies based on the search results:

This table provides researchers with a quick reference for selecting the appropriate antibody based on their specific experimental requirements, model organism, and intended applications .

How should CLASP1 antibodies be stored and handled to maintain optimal performance?

Proper storage and handling of CLASP1 antibodies is crucial for maintaining their performance over time:

• Long-term storage: Store antibodies at -20°C for optimal long-term stability. According to product information, CLASP1 antibodies can be stored at -20°C for up to one year .

• Short-term storage: For frequent use over shorter periods (up to one month), antibodies can be stored at 4°C .

• Avoid freeze-thaw cycles: Repeated freeze-thaw cycles can significantly reduce antibody activity. Aliquot the antibody into smaller volumes upon first use to minimize freeze-thaw cycles .

• Buffer composition: Most CLASP1 antibodies are provided in a stabilizing buffer containing components such as PBS, glycerol, BSA, and sodium azide. The Boster antibody, for example, is formulated in PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide . This formulation helps maintain antibody stability.

• Working dilutions: Prepare working dilutions fresh on the day of the experiment rather than storing diluted antibody for extended periods.

• Contamination prevention: Use sterile technique when handling antibodies to prevent microbial contamination, which can degrade the antibody and introduce experimental artifacts.

• Temperature transitions: Allow refrigerated antibodies to equilibrate to room temperature before opening to prevent condensation, which could dilute or contaminate the antibody.

Proper documentation of antibody performance over time can help identify potential degradation issues and ensure experimental reproducibility.

How can I design experiments to distinguish between CLASP1 and CLASP2 functions?

Designing experiments to distinguish between CLASP1 and CLASP2 functions requires careful consideration of their similarities and differences:

• RNA interference approaches: Use specific siRNAs targeting either CLASP1 or CLASP2 individually, as well as combined knockdown. Previous studies have successfully used pairs of siRNAs (CLASP1#A/B and CLASP2#A/B) to achieve approximately 70% reduction in protein levels . This approach allows for comparing phenotypes between single and double knockdowns to identify unique and redundant functions.

• Rescue experiments: After knockdown, perform rescue experiments with siRNA-resistant constructs expressing either CLASP1 or CLASP2 to confirm specificity and test functional redundancy.

• Domain-specific analysis: Create chimeric proteins or domain deletions to identify which protein domains are responsible for specific functions or localizations.

• Cell type-specific analysis: Test CLASP1 and CLASP2 functions across different cell types, as their roles may vary. For example, in neuronal cells, CLASP1 and CLASP2 show different effects on neurite outgrowth depending on the neuronal population .

• Protein-protein interaction studies: Investigate binding partners specific to each CLASP protein. While they share some interactors like CLIP-115 and CLIP-170, they may also have unique binding partners .

• High-resolution localization studies: Use super-resolution microscopy to determine if CLASP1 and CLASP2 have distinct subcellular localizations, which might indicate non-overlapping functions.

When interpreting results, consider that CLASP1 and CLASP2 are coexpressed in many cell types , so complete functional separation may be challenging to achieve.

What controls should be included when using CLASP1 antibodies for critical research applications?

For rigorous research using CLASP1 antibodies, include these essential controls:

• Positive controls:

  • Cell lines with known CLASP1 expression (SH-SY5Y, Jurkat, HepG2, MCF7)

  • Mouse or rat brain tissue for neuronal studies

  • Overexpression systems with tagged CLASP1 constructs

• Negative controls:

  • Peptide competition: Pre-incubate the antibody with the immunizing peptide to block specific binding

  • Secondary antibody only: Omit primary antibody to assess non-specific binding of the secondary antibody

  • CLASP1 knockdown: Use validated siRNAs to reduce CLASP1 expression

  • Isotype control: Use normal rabbit IgG at the same concentration as the CLASP1 antibody

• Cross-reactivity controls:

  • Test the antibody against overexpressed CLASP2 to assess potential cross-reactivity

  • Include CLASP2 knockdown samples to ensure signals are CLASP1-specific

• Application-specific controls:

  • For Western blot: Include molecular weight markers and loading controls

  • For IHC/IF: Include autofluorescence controls and counterstains for structural context

  • For co-localization studies: Include single-stained samples to assess bleed-through

• Validation across methods: Confirm findings using multiple detection methods (e.g., if you see localization by IF, confirm with fractionation and Western blot)

Comprehensive controls not only validate your findings but also help troubleshoot when experiments don't yield expected results.

How can researchers stay updated on new findings about CLASP1 function and antibody technologies?

To stay current with CLASP1 research and antibody technologies:

• Set up publication alerts: Create alerts in PubMed, Google Scholar, and research databases using keywords like "CLASP1," "cytoplasmic linker associated protein 1," and "microtubule plus-end tracking proteins."

• Join relevant scientific societies: Organizations focused on cell biology, cytoskeleton research, and neuroscience often share updates on proteins like CLASP1. The American Society for Cell Biology (ASCB) and the International Society for Neurochemistry frequently cover such topics.

• Attend specialized conferences: Conferences on cytoskeleton dynamics, cell division, or neurodevelopment often feature the latest CLASP1 research. The ASCB annual meeting and Gordon Research Conferences on related topics are valuable resources.

• Engage with antibody validation initiatives: Programs like the Antibody Validation Initiative provide updated information on antibody specificity and applications.

• Collaborate with leading laboratories: Establish collaborations with groups actively researching CLASP proteins to access specialized reagents and techniques.

• Monitor antibody resource databases: Repositories like Antibodypedia and the Antibody Registry provide user reviews and validation data for specific antibodies.

• Participate in method-sharing platforms: Sites like protocols.io allow researchers to share optimized protocols for CLASP1 detection across various applications.

Regular engagement with these resources ensures that your CLASP1 research employs the most current knowledge and methodologies, enhancing the reliability and impact of your findings.

What emerging applications or techniques might enhance CLASP1 antibody-based research in the future?

Several emerging technologies hold promise for advancing CLASP1 antibody-based research:

• Proximity labeling approaches: Techniques like BioID or APEX2 combined with CLASP1 antibodies could reveal transient interaction partners at specific cellular locations, particularly at dynamic structures like microtubule plus-ends.

• Single-molecule imaging technologies: Super-resolution techniques combined with specific CLASP1 antibodies will enable nanoscale visualization of CLASP1 dynamics at microtubule plus-ends and other cellular structures.

• Antibody engineering: Recombinant antibody technologies producing smaller formats like nanobodies or single-chain variable fragments (scFvs) against CLASP1 could enable live-cell imaging with reduced interference.

• Spatially-resolved proteomics: Techniques like imaging mass cytometry combined with CLASP1 antibodies could reveal spatial distribution of CLASP1 in tissues with unprecedented resolution.

• CRISPR epitope tagging: Endogenous tagging of CLASP1 using CRISPR-Cas9 combined with well-characterized tag-specific antibodies will enable more physiological studies of CLASP1 function.

• Automated high-content screening: Machine learning-powered image analysis of CLASP1 antibody staining could identify subtle phenotypes across large datasets from genetic or drug screens.

• Cryo-electron tomography with immunogold labeling: This approach could reveal the 3D organization of CLASP1 at microtubule plus-ends and kinetochores at nanometer resolution.

• Single-cell Western blot technologies: These could enable analysis of CLASP1 expression and modification states in rare cell populations or at specific cell cycle stages.