CLASP Antibody

What are CLASP proteins and why are they important targets for antibody research?

CLASP1 and CLASP2 (Cytoplasmic Linker Associated Proteins) are microtubule plus-end tracking proteins that promote the stabilization of dynamic microtubules . These proteins play critical roles in regulating the density, length distribution, and stability of interphase microtubules . CLASP2 is specifically involved in the nucleation of noncentrosomal microtubules originating from the trans-Golgi network and is required for the polarization of cytoplasmic microtubule arrays in migrating cells . At the molecular level, CLASP proteins may enhance the frequency of rescue of depolymerizing microtubules by attaching their plus-ends to cortical platforms composed of ERC1 and PHLDB2 . Understanding these functions makes CLASP proteins important targets for antibody-based research in cell division, migration, and cytoskeletal organization studies.

What types of CLASP antibodies are available for research applications?

Several types of CLASP antibodies are available for research applications:

These antibodies recognize endogenous CLASP proteins with molecular weights of approximately 160 kD, corresponding to the predicted size of CLASP1/2α isoforms .

How can I validate the specificity of CLASP antibodies in my experimental system?

Validating CLASP antibodies involves multiple approaches to ensure specificity:

• siRNA knockdown control: Compare antibody signals in control cells versus CLASP-depleted cells (via siRNA). After CLASP1+2 siRNA treatment, all CLASP-specific signals should be significantly reduced in immunofluorescence and Western blot analyses .

• Cross-reactivity assessment: Test antibodies against overexpressed GFP-tagged CLASP1 and CLASP2 to quantify any cross-reactivity between the two proteins .

• FACS analysis: Flow cytometry after staining with CLASP antibodies can verify if knockdown cells represent a homogeneous population with reduced antibody signal .

• Signal localization verification: Confirm that antibodies detect CLASP at expected subcellular locations, such as MT tips, Golgi, and centrosomes .

• Multiple antibody comparison: Use multiple antibodies against different epitopes of the same protein to confirm consistent results.

How can CLASP antibodies be used to study microtubule dynamics and organization?

CLASP antibodies are powerful tools for investigating microtubule dynamics and organization:

• Quantitative immunofluorescence: CLASP antibodies can be used to analyze how CLASP depletion alters microtubule organization. Studies have shown that after CLASP knockdown, microtubules often orient along the cell margin, whereas in control cells most microtubules are perpendicular to the cell edge .

• Microtubule angle analysis: Using CLASP antibodies alongside tubulin staining allows researchers to quantify the angles between microtubules and the cell margin. After CLASP1+2 knockdown, the number of microtubules deviating by >45° from the cell radius increased by a factor of 2.6, demonstrating CLASPs' role in maintaining proper microtubule orientation .

• Membrane dynamics studies: CLASP antibodies can help investigate the relationship between microtubule organization and membrane dynamics. Research has shown that the rate of retrograde membrane flow was reduced almost by half in CLASP1+2 siRNA-treated cells compared with control .

• Rescue frequency measurement: By combining CLASP immunolabeling with live-cell imaging of microtubule plus-ends, researchers can correlate CLASP levels with microtubule rescue frequencies at specific cellular locations.

What are the optimal conditions for using CLASP2 antibodies in immunohistochemistry?

For optimal results with CLASP2 antibodies in immunohistochemistry on paraffin sections:

These conditions have been validated for mouse brain tissue sections and may require optimization for other tissue types or experimental systems .

How do I design experiments to distinguish between CLASP1 and CLASP2 functions given their functional redundancy?

Designing experiments to distinguish between CLASP1 and CLASP2 functions requires sophisticated approaches:

• Selective knockdown strategy: Use multiple siRNAs specific for each CLASP (e.g., CLASP1#A, CLASP1#B, CLASP2#A, CLASP2#B) individually and in combination to achieve varying levels of depletion .

• Quantitative phenotype analysis: Compare phenotypes between single and double knockdowns to identify subtle differences in function. For example, analyze MT density, orientation, and stability parameters .

• Rescue experiments: Reintroduce siRNA-resistant versions of either CLASP1 or CLASP2 into double-knockdown cells to determine which functions can be rescued by each protein.

• Domain swap experiments: Create chimeric proteins containing domains from both CLASPs to identify regions responsible for unique functions.

• Tissue-specific analysis: Examine CLASP localization and function in different cell types where one may predominate over the other.

The redundancy between CLASPs has been established in several studies, with both proteins playing overlapping roles in regulating microtubule density, length distribution, and stability .

What is the "clasping antibody" technology and how does it differ from conventional antibodies?

The clasping antibody technology represents a paradigm-shifting approach to antibody-antigen recognition:

In conventional antibodies, each antigen-binding fragment (Fab) functions as an autonomous unit for antigen recognition with a 1:1 stoichiometry between antigen and Fab . In contrast, clasping antibodies employ a previously unidentified mode of antibody-antigen recognition where two antigen-binding sites cooperatively clasp a single antigen .

The key differences include:

This unique binding mode enables clasping antibodies to achieve extraordinarily high specificity for challenging targets like histone post-translational modifications .

What are the key advantages of clasping antibodies for epigenetic research?

Clasping antibodies offer several significant advantages for epigenetic research:

• Superior specificity: The extensive interaction surface created by antigen clasping enables exquisite specificity for histone post-translational modifications. In head-to-head comparisons, clasping antibodies exhibited superior specificity to widely used conventional antibodies in chromatin immunoprecipitation (ChIP) applications .

• High affinity binding: Clasping antibodies achieve tight binding in the low nanomolar to sub-nanomolar affinity range .

• Minimal lot-to-lot variation: Being recombinant antibodies, clasping antibodies have minimal lot-to-lot variation compared to traditional polyclonal and monoclonal antibodies produced through animal immunization .

• Recognition of challenging epitopes: The unique binding mode makes clasping antibodies particularly effective for challenging targets like site-specific post-translational modifications (PTMs) .

• Precise epitope recognition: For histone modifications, the clasping antibodies use an unusually extensive aromatic cage in one Fab to bind the trimethylated lysine and a pocket in the other Fab to recognize the histone N terminus, enabling highly specific recognition of the exact modification state .

These advantages make clasping antibodies particularly valuable for epigenetic research where antibody specificity has been a persistent challenge affecting data reproducibility.

How does the structural basis of antigen clasping enable high-specificity recognition?

The structural basis of antigen clasping reveals several key features that enable high-specificity recognition:

• Cooperative recognition: Two antigen-binding sites form a head-to-head dimer and cooperatively recognize the antigen in the dimer interface .

• Expansive binding interface: The clasping creates extensive interactions between the antibody and antigen. The interfaces (1,177 Ų for the H3K9me3 peptide and 987 Ų for the H3K4me3 peptide) are nearly twice as large as typical peptide-protein binding interfaces .

• Multiple CDR utilization: Clasping antibodies utilize most or all of their CDRs for antigen recognition. Anti-H3K9me3 clasping antibody (309M3-B) used a total of 8 CDRs, while anti-H3K4me3 (304M3-B) used all 12 CDRs available in the Fab dimer .

• Specialized binding pockets: For histone modifications, one Fab contributes an extensive aromatic cage that binds trimethylated lysine, while the other Fab provides a pocket that recognizes the histone N terminus. This dual recognition ensures exceptionally high specificity .

• Flat binding topography: Unlike typical anti-peptide antibodies with deep clefts created by long CDRs, clasping antibodies have flat antigen-binding sites with short CDRs, similar to antibodies targeting large spherical antigens like structured proteins .

This structural arrangement enables clasping antibodies to achieve both high specificity and high affinity, making them particularly valuable for recognizing challenging epitopes.

How are clasping antibodies generated and optimized for targeting specific histone modifications?

The generation of clasping antibodies involves a sophisticated process:

• Initial clone identification: Researchers identified an antibody (clone 4-5) that weakly but specifically recognized trimethyl lysine from a naive human antibody library .

• Directed evolution: Using this initial clone, they generated a single chain Fv (scFv) phage-display library where they diversified a subset of CDR residues .

• Stringent selection: Multiple rounds of stringent selection for specific binding to histone peptides were performed to isolate new antibodies with high specificity for targets like H3K9me3 and H3K4me3 .

• Rational design approach: A binding mode-guided approach was used to design antibodies that could utilize the antigen-clasping mechanism .

• Synthetic platform development: Researchers developed a synthetic yeast-display system where the single-chain variable fragment (scFv) enhancer arm is tethered to another PTM-specific scFv arm via a long linker to facilitate intra-molecular dimerization .

• Novel antibody format: A specialized IgG-like antibody format with a long linker between the antigen-binding module and the Fc region was engineered to facilitate antigen clasping and achieve both high specificity and high potency .

This systematic approach yielded antibodies like 309M3-B (anti-H3K9me3) and 304M3-B (anti-H3K4me3) with extraordinary specificity for their target modifications .

What experimental evidence confirms the clasping binding mode in solution?

The clasping binding mode was confirmed through multiple experimental approaches:

• Size-exclusion chromatography: The sizes of antibody-peptide complexes were approximately twice as large as the antibodies alone, indicating dimerization upon antigen binding .

• Dynamic light scattering: This technique provided additional confirmation of the size shift observed in the antibody-peptide complexes .

• Stoichiometry analysis: A 2:1 molar ratio of antibody to peptide was sufficient to fully induce the size shift of 309M3-B (anti-H3K9me3), whereas a 1:1 ratio was required for 304M3-B (anti-H3K4me3). These binding stoichiometries in solution were consistent with those observed in crystal structures .

• Control experiments: Trimethyl lysine alone did not induce the size shift of 309M3-B, confirming that the complete peptide epitope was required for clasping .

• Crystallographic analysis: Crystal structures revealed that two antigen-binding sites of these antibodies form a head-to-head dimer and cooperatively recognize the antigen in the dimer interface .

These multiple lines of evidence conclusively demonstrated antigen-induced dimerization of clasping antibodies, confirming this novel mode of antibody-antigen recognition.

What are the methodological considerations for implementing clasping antibodies in ChIP and other epigenetic applications?

When implementing clasping antibodies in chromatin immunoprecipitation (ChIP) and other epigenetic applications, researchers should consider these methodological aspects:

• Antibody concentration optimization: The unique binding mode of clasping antibodies may require different optimal concentrations compared to conventional antibodies. Titration experiments should be performed to determine ideal antibody amounts.

• Incubation conditions: The formation of the antibody-antigen clasping complex may have different kinetics than conventional antibody binding, potentially requiring adjusted incubation times or temperatures.

• Crosslinking considerations: For ChIP applications, the larger binding interface created by clasping antibodies may be differently affected by formaldehyde crosslinking. Optimization of crosslinking conditions might be necessary.

• Buffer composition: Ensure that buffer conditions are compatible with the clasping binding mode, which involves antibody dimerization upon antigen binding.

• Validation controls: Include appropriate controls such as input samples, IgG controls, and known positive/negative regions for the histone modification of interest.

• Data analysis awareness: The superior specificity of clasping antibodies may reveal more precise patterns of histone modifications than conventional antibodies, potentially yielding results that differ from previous studies using less specific reagents .

Proper implementation of these considerations will help researchers take full advantage of the superior specificity offered by clasping antibodies in epigenetic research applications.

What roles do CLASP proteins play in disease processes and how can CLASP antibodies facilitate disease research?

While not extensively covered in the provided search results, CLASP proteins have important implications in disease processes through their roles in microtubule dynamics, cell division, and migration:

• Cancer research: CLASP antibodies can help investigate how alterations in microtubule dynamics contribute to cancer cell migration and invasion. CLASP2 acts as a mediator of ERBB2-dependent stabilization of microtubules at the cell cortex , potentially linking it to ERBB2-positive breast cancers.

• Neurological disorders: CLASPs play roles in neuronal development and function through their effects on microtubule stability. CLASP antibodies can be used to study changes in neuronal microtubule organization in various neurological conditions.

• Cell division abnormalities: Since CLASPs perform essential stabilizing functions at the kinetochore during mitosis , CLASP antibodies can help investigate mitotic defects in conditions characterized by chromosomal instability.

• DNA damage response: Claspin (not to be confused with CLASPs) functions in the ATR-Chk1 pathway essential for DNA damage response . Antibodies against Claspin can be valuable for studying DNA damage response pathways in cancer and aging.

CLASP antibodies facilitate disease research by enabling precise visualization of protein localization, quantification of expression levels, and analysis of post-translational modifications in normal versus diseased tissues.

How can we optimize CLASP antibody-based immunohistochemistry for diagnostic applications?

Optimizing CLASP antibody-based immunohistochemistry for potential diagnostic applications requires:

• Tissue-specific protocol optimization: As demonstrated with anti-CLASP2 [KT68] on mouse brain tissue , each tissue type may require specific conditions for optimal staining. Parameters to optimize include:

  • Antigen retrieval method (heat-mediated vs. enzymatic)

  • Buffer composition and pH (e.g., sodium citrate buffer at pH6)

  • Antibody concentration and incubation time

  • Detection system (e.g., HRP conjugated compact polymer system)

• Validation across multiple samples: Establish reproducibility across samples from different patients/sources and create reference standards for staining patterns and intensities.

• Automated staining platforms: Adapt protocols to automated immunohistochemistry platforms to improve reproducibility for clinical applications.

• Multiplex staining approaches: Develop protocols for simultaneous detection of CLASPs alongside other biomarkers to increase diagnostic value.

• Digital pathology integration: Implement quantitative image analysis of CLASP immunohistochemistry for objective assessment and potential correlation with clinical outcomes.

These optimizations could potentially position CLASP antibody-based immunohistochemistry as a valuable tool in research and diagnostic applications.