CLASP2 Antibody

What is CLASP2 and what cellular functions does it regulate?

CLASP2, also known as cytoplasmic linker associated protein 2, KIAA0627, or hOrbit2, is a 1,294 amino acid protein characterized by five HEAT repeats. It primarily localizes to the cytoplasm, cytoskeleton, kinetochore, and Golgi apparatus, with particularly prominent expression in brain tissue. CLASP2 functions as a microtubule plus-end tracking protein (+TIP) that regulates dynamic microtubule stability and ensures proper polarization of cytoplasmic microtubule arrays in migrating cells . CLASP2 plays critical roles in maintaining kinetochore stability and ensuring accurate chromosomal alignment during cell division. Research has shown that CLASP1 and CLASP2 play redundant roles in regulating the density, length distribution, and stability of interphase microtubules .

What detection methods work best with CLASP2 antibodies?

CLASP2 antibodies, such as the F-3 mouse monoclonal IgG1 kappa light chain antibody, can effectively detect CLASP2 across multiple species (mouse, rat, and human) using several techniques. The most reliable detection methods include western blotting (WB), immunoprecipitation (IP), immunofluorescence (IF), immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA) . For optimal results in immunofluorescence applications, researchers should note that CLASP2 typically decorates the Golgi apparatus, centrosome, and microtubule plus ends, producing specific staining patterns that colocalize with other +TIPs like EB1, CLIP-170, and p150 .

What are the key interacting partners of CLASP2?

CLASP2 interacts with several key proteins as part of its function in microtubule regulation. Notable interaction partners include EB1, EB3, ELKS, and CLIP-115, which collectively contribute to microtubule stabilization . Recent affinity purification coupled with mass spectrometry (AP-MS) experiments have further expanded our understanding of the CLASP2 interactome. In 3T3-L1 adipocytes, CLASP2 has been found to co-immunoprecipitate with the novel protein SOGA1, the microtubule-associated protein kinase MARK2, and the microtubule/actin-regulating protein G2L1 . Additionally, structural studies have identified a consensus sequence for CLASP2 binding in proteins like CLIP170 and LL5β, with research pinpointing the minimal CLASP-binding regions in these proteins .

How do post-translational modifications affect CLASP2 function?

CLASP2 undergoes phosphorylation by glycogen synthase kinase 3 beta (GSK3β), a modification that has been shown to reduce its microtubule-binding capacity . In the context of insulin signaling, CLASP2 undergoes insulin-stimulated phosphorylation, which leads to its co-localization with reorganized actin and GLUT4 at the plasma membrane . These post-translational modifications represent important regulatory mechanisms that modulate CLASP2's interactions with microtubules and other cellular components, thereby influencing its functional roles in various cellular processes.

How do the distinct TOG domains of CLASP2 contribute differently to its function?

CLASP2 contains two distinct TOG (tumor overexpressed gene) domains that exhibit different affinities for tubulin and microtubules. Structural analyses have revealed that the two TOG domains (TOG2 and TOG3) have unique conformational features that affect their interactions with tubulin. When comparing the structures of the multiple HEAT repeats (HRs) between TOGs, researchers found that HRs including HR3 aligned poorly, whereas HRs without HR3 showed lower RMS deviation values, suggesting structural rearrangement at HR3 of CLASP2-TOG2 .

The second HR triad (HR4-6) of human CLASP2-TOG2 shows a dramatic shift, creating a convex arch on the tubulin-binding surface. In contrast, mouse CLASP2-TOG3 forms a relatively flat plane on its tubulin-binding surface, though the paddle structure curves around an axis perpendicular to this surface . Mutation studies have demonstrated that alterations to either TOG domain or EB1-binding sites decrease the rescue frequency of microtubules in vivo, indicating that both TOGs and EB1 binding are essential for the full rescue activity of CLASP2 .

What methodological considerations are important when performing CLASP2 knockdown experiments?

When conducting CLASP2 knockdown experiments, researchers have successfully employed small interfering RNAs (siRNAs) specific for CLASP2. Studies have utilized multiple siRNAs (e.g., CLASP2#A and CLASP2#B) to ensure specificity and reproducibility. For optimal knockdown, transfection protocols typically involve a three-day incubation period, resulting in approximately 70% reduction in CLASP2 levels, which can be considered a hypomorphic state .

Verification of knockdown efficiency should be performed using both Western blotting and immunofluorescence. Researchers should be aware that partial down-regulation of CLASPs individually may have subtle phenotypes, while simultaneous knockdown of CLASP1 and CLASP2 produces more pronounced effects, including a 40% reduction in acetylated tubulin levels, indicating decreased microtubule stability . Additionally, FACS analysis after staining with CLASP1 and CLASP2 antibodies can be used to confirm homogeneity in knockdown cell populations .

How can researchers accurately distinguish between CLASP1 and CLASP2 in experimental settings?

Distinguishing between CLASP1 and CLASP2 can be challenging due to their structural similarities and potential cross-reactivity of antibodies. Researchers have developed specific antibodies for each protein, though some cross-reactivity often remains. For instance, antibodies #402 and #2292 strongly react with GFP-CLASP1α but display some cross-reactivity with GFP-CLASP2α. Similarly, antibody #2358 strongly reacts with GFP-CLASP2α and cross-reacts to some extent with GFP-CLASP1α .

To achieve more specific detection, researchers should carefully select antibodies based on their epitope recognition sites and validate their specificity using overexpression and knockdown approaches. When performing knockdown experiments, using siRNAs specific to each CLASP variant can help delineate their individual functions. Western blotting can further confirm antibody specificity, as both CLASP1 and CLASP2α isoforms appear as proteins of approximately 160 kD .

What techniques are most effective for studying CLASP2 protein interactions in vitro?

Several techniques have proven effective for studying CLASP2 protein interactions. Analytical size exclusion chromatography (aSEC) and isothermal titration calorimetry (ITC)-based measurements have been successfully used to map regions critical for binding between CLASP2's TOG4 domain and interacting proteins like CLIP170 and LL5β .

For broader interactome studies, affinity purification coupled with mass spectrometry (AP-MS) combined with label-free quantitative proteomics has been effectively employed. This approach typically involves:

• Incubating cell lysate (3.5–5 mg) with 5 μg of specific antibodies conjugated to protein A or protein G-agarose beads for 3 hours at 4°C with gentle rotation

• Washing immunoprecipitates three times with ice-cold PBS

• Eluting bound proteins by heating at 95°C in SDS sample loading buffer

• Performing a second elution to maximize protein recovery

• Analyzing combined eluates by SDS-PAGE followed by either Coomassie staining or Western blotting

Data analysis tools like Significance Analysis of Interactome (SAINT) can then be used to identify significant protein interactions and build comprehensive protein networks .

What controls should be included when using CLASP2 antibodies for immunofluorescence?

When performing immunofluorescence with CLASP2 antibodies, several critical controls should be included to ensure specificity and reliability of results. First, researchers should include a knockdown control using CLASP2-specific siRNAs to verify the specificity of the observed staining pattern. After CLASP1+2 siRNA treatment, all CLASP-specific signals should be significantly reduced, with diminished staining at microtubule tips, Golgi, and centrosome .

Second, positive controls using cells overexpressing tagged CLASP2 (e.g., GFP-CLASP2α) can help confirm antibody specificity and determine optimal antibody concentrations. Third, researchers should verify colocalization with known CLASP2 interacting partners, such as EB1, CLIP-170, and p150, particularly at microtubule plus ends . Finally, including negative controls (secondary antibody only) and IgG isotype controls will help identify any non-specific background staining.

How can researchers optimize Western blotting protocols for CLASP2 detection?

For optimal CLASP2 detection via Western blotting, researchers should carefully consider several technical aspects:

• Sample preparation: Cell lysates should be prepared using protocols that preserve CLASP2 integrity, such as lysis in buffer containing protease inhibitors and phosphatase inhibitors if phosphorylated forms are of interest.

• Protein loading: Load 15-30 μg of total protein per lane for cell lysates, as CLASP2 is typically expressed at moderate levels.

• Gel percentage: Use 7.5% or 10% SDS-PAGE gels to properly resolve the high molecular weight CLASP2 protein (approximately 160 kD) .

• Transfer conditions: Employ longer transfer times (60-90 minutes at 100V) or overnight transfers at lower voltage to ensure efficient transfer of the large CLASP2 protein.

• Blocking conditions: Block membranes with 5% nonfat dry milk in Tris-buffered saline with 0.2% Tween-20 for 1 hour at room temperature .

• Antibody dilutions: Primary CLASP2 antibodies should typically be used at 1:500-1:1000 dilutions, while secondary antibodies (goat anti-mouse or donkey anti-rabbit) conjugated to horseradish peroxidase are effective at 1:1500 dilutions .

• Detection method: Enhanced chemiluminescence (ECL) provides sufficient sensitivity for CLASP2 detection in most experimental settings .

What are the recommended protocols for immunoprecipitation of CLASP2?

For successful immunoprecipitation of CLASP2, researchers should follow these recommended steps:

• Prepare cell lysates from approximately one 150 mm tissue culture dish (yielding 3.5-5 mg total protein) using appropriate lysis buffers that maintain protein-protein interactions.

• Conjugate 5 μg of specific CLASP2 antibodies to 25 μl protein A or protein G-agarose beads.

• Incubate the cell lysate with antibody-conjugated beads for 3 hours at 4°C with gentle rotation to allow efficient capture of CLASP2 and its interacting partners.

• Wash the immunoprecipitates thoroughly (at least three times) with 1 ml of ice-cold PBS to remove non-specifically bound proteins.

• Elute bound proteins by heating at 95°C for 4 minutes in SDS sample loading buffer (containing 4% SDS, 0.0625 M Tris-HCl, 10% glycerol, 0.02% bromphenol blue, 8 M Urea).

• Perform a second elution on the antibody/beads to maximize protein recovery and combine both eluates for analysis.

• Analyze the immunoprecipitated proteins by SDS-PAGE followed by Western blotting or mass spectrometry depending on experimental goals .

Why might researchers observe cross-reactivity between CLASP1 and CLASP2 antibodies?

Cross-reactivity between CLASP1 and CLASP2 antibodies is a common challenge due to the high sequence homology between these proteins. Studies have shown that antibodies raised against CLASP1 (#402 and #2292) strongly react with GFP-CLASP1α but also display some cross-reactivity with GFP-CLASP2α. Similarly, antibodies against CLASP2 (#2358) strongly react with GFP-CLASP2α but cross-react to some extent with GFP-CLASP1α .

This cross-reactivity likely stems from conserved epitopes within the protein structures. To minimize this issue, researchers should:

• Carefully select antibodies based on regions with greater sequence divergence between CLASP1 and CLASP2

• Validate antibody specificity using overexpression systems with tagged versions of each protein

• Employ knockdown approaches (using specific siRNAs for each protein) to confirm antibody specificity

• Consider using complementary approaches, such as mass spectrometry, to definitively identify the protein of interest

• When possible, use antibodies raised against different epitopes for confirmation of results

How can researchers address inconsistent results when studying CLASP2 and tubulin interactions?

Inconsistent results when studying CLASP2-tubulin interactions may stem from several factors. For instance, contradictory data have been observed regarding CLASP2-TOGs' affinity for tubulin dimers in comparison to in vitro MT assembly analysis of Drosophila CLASP homolog MAST .

To address these inconsistencies, researchers should:

• Consider species-specific differences in CLASP proteins and their interactions with tubulin

• Account for post-translational modifications, particularly phosphorylation by GSK3β, which can significantly reduce CLASP2's microtubule-binding capacity

• Ensure consistent experimental conditions regarding temperature, pH, and salt concentrations, as these factors can affect protein-protein interactions

• Use multiple complementary approaches to study these interactions (e.g., co-sedimentation assays, TIRF microscopy, and immunoprecipitation)

• Employ both in vitro reconstitution systems and cellular models to validate findings

• Consider the specific isoforms of CLASP2 being studied, as alternative splicing generates multiple variants (e.g., beta and gamma) with potentially diverse functional properties