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CLASP2 · Western blot design guide
Design a Western Blot for CLASP2
Real validated CLASP2 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-CLASP2 WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Last reviewed: May 2026 · Scientific review: Boster Bio technical team
CLASP2 Western Blot Experimental Design Guide
Expected bands, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before running
| Expected band | ~141.1 kDa | |
| Observed band | ~165 kDa | |
| Gel | 8–10% | |
| Negative control | siRNA / KO lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated | |
| Caveat | Phosphorylation-induced shift | |
| Regulation | LPS-induced | |
| Isoform | 3 isoform(s) |
Section 1
Real Curated CLASP2 Western Blot Protocols
Literature-validated Western blot parameters for CLASP2 — gel percentage, transfer, blocking, antibody incubation and detection, extracted from published methods.
| Sample / lysate | human HEK293 , Lane 2: human SH-SY5Y , Lane 3: human HL-60 , Lane 4: rat brain , Lane 5: mouse brain . After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-CLASP2 antigen affinity purified polyclonal antibody (Catalog # A03579-1) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for CLASP2 at approximately 165 kDa. The expected band size for CLASP2 is at 141 kDa |
| Gel % | 8–10% |
| Load | 30 ug |
| Transfer | nitrocellulose membrane, 150 mA, 50–90 min |
| Membrane | nitrocellulose |
| Blocking | 5% non-fat milk / TBS, 1.5 h RT |
| Primary antibody | 0.5 µg/mL |
| Primary incubation | overnight at 4 °C |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 |
| Wash | TBS-0.1% Tween, 3 × 5 min |
| Detection | ECL |
| Exposure / imaging | Tanon 5200 |
| Observed band | 165 kDa |
Section 2
What Is the Expected CLASP2 Western Blot Band Size?
CLASP2 has a 141 kDa predicted backbone but typically runs at ~165 kDa on Western blot due to extensive phosphorylation and its large, coiled-coil-rich cytoskeletal structure.
| major band at ~165 kDa in whole-cell lysate | the expected full-length CLASP2 signal, running above its 141 kDa predicted mass due to extensive phosphorylation and the protein's elongated, coiled-coil-rich structure |
| band positioned visibly higher than the 141 kDa calculated mass | extensive phosphorylation across 28 or more modified serine/threonine residues alters SDS-PAGE mobility without adding covalent mass the way glycosylation would |
| multiple bands at distinct molecular weights | reflects expression of more than one of the three annotated CLASP2 splice isoforms within the same lysate |
| diffuse or slightly fuzzy band rather than one crisp line | heterogeneous phosphorylation occupancy across the cell population produces a narrow spread of migration rates around the ~165 kDa band |
| band unchanged between reducing and non-reducing conditions | CLASP2 has no annotated disulfide bonds, so it migrates as a monomer regardless of reducing agent |
💡Expected CLASP2 appearanceExpect a single dominant CLASP2 band at approximately 165 kDa in whole-cell lysate, running above its 141 kDa predicted mass due to extensive phosphorylation and the protein's large, coiled-coil-rich structure rather than glycosylation or proteolytic processing.
| Predicted mass (UniProt) | calculated backbone mass of 141.1 kDa across 1294 residues sets the theoretical baseline before any modification is considered |
| Extensive phosphorylation (28 modified residues) | multiple phosphoserine sites shift apparent migration upward, contributing to the ~165 kDa observed band rather than the 141 kDa predicted mass |
| Alternative splicing (isoforms 1, 2, 3) | different isoforms can appear as additional bands at qualitatively different apparent sizes depending on which exons are included |
| No annotated disulfide bonds | band position is unaffected by reducing versus non-reducing SDS-PAGE conditions since CLASP2 does not form inter-chain disulfide-linked complexes |
| No signal peptide or propeptide | no proteolytic maturation step is expected, so a smaller cleaved fragment should not appear below the full-length band |
| Situation | Likely cause | Next action |
|---|---|---|
| Band higher than expected | extensive phosphorylation and the protein's elongated coiled-coil conformation cause the native band to run above the 141 kDa predicted mass | reference the empirical ~165 kDa band rather than the calculated mass and treat this shift as expected, not a technical artifact |
| Multiple bands | the three annotated CLASP2 splice isoforms can produce additional bands at different apparent sizes | check which isoform(s) the tissue or cell line is known to express before treating extra bands as nonspecific |
| Broad smear instead of sharp band | heterogeneous phosphorylation occupancy across the cell population broadens the migration profile | resolve on a lower-percentage or gradient gel and keep phosphatase inhibitors consistent across samples for cleaner comparison |
| Weak or no signal | CLASP2 is a large cytoskeleton-, centrosome-, and kinetochore-associated protein that can partition into insoluble cellular fractions and be under-extracted by standard lysis buffers | use a stronger lysis buffer with mechanical disruption to solubilize cytoskeletal-bound protein and load more total protein |
| Fragments below expected size | large multi-domain proteins like CLASP2 are prone to proteolytic degradation during lysate preparation | include protease inhibitors and keep samples cold throughout lysis and loading to minimize degradation fragments |
Experiment evidence
Western blot images showing CLASP2 band
A03579-1 · CLASP2
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for CLASP2 Western blot
🧪For positive controls for CLASP2 in Western blot, you can use HeLa cell lysate, since CLASP2 is a broadly expressed cytoskeletal/centrosome-kinetochore protein present in essentially all dividing cells and no Human Protein Atlas tissue expression data are available to identify a more specific source.
Positive control: HeLa cells
Negative control: ubiquitously expressed; use siRNA knockdown or KO line
Loading controls: Run GAPDH and β-actin antibodies alongside a total-protein stain (e.g., stain-free gel, Ponceau S, or REVERT) to confirm equal loading and transfer.
⚠️Feasibility: No HPA expression data exist for CLASP2, and as a cytoskeletal/kinetochore-associated protein it is likely expressed across most cell types, so a clean negative-tissue control is unlikely and antibody specificity should instead be validated with CLASP2 siRNA knockdown or a CRISPR knockout line.
Comprehensive Human Protein Atlas IHC scoring per tissue. Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|
Section 3
Advanced CLASP2 Western Blot Tips
Deeper troubleshooting and optimisation questions for CLASP2, answered from its protein features.
Why does CLASP2 run at ~165 kDa instead of predicted 141 kDa?
CLASP2 carries 28 modified residues (mostly phosphorylation) reported in UniProt; heavy phosphorylation and its large, repeat-containing structure commonly retard SDS-PAGE mobility, producing an apparent mass above the calculated 141.1 kDa. Confirm identity with a positive control lysate and, if available, phosphatase treatment to test whether the shift shrinks toward predicted mass.
Which CLASP2 isoform will my antibody detect?
UniProt lists three CLASP2 isoforms (1, 2, 3) generated by alternative splicing. Check the immunogen region against each isoform sequence before interpreting band number; an antibody raised against a common region can show one band, while an N- or C-terminal-specific antibody may only detect a subset, causing apparent isoform-specific bands or missing bands.
Does CLASP2 phosphorylation change with cell cycle stage?
CLASP2 is annotated as a phosphoprotein with 28 modified residues and keywords for Cell cycle, Mitosis, Centromere, and Kinetochore, reflecting its role in mitotic microtubule and kinetochore regulation. Expect phosphorylation-dependent mobility or band intensity shifts in mitotic versus interphase lysates; synchronize cells if comparing phospho-status.
What transfer method to use for CLASP2 Western blot?
At ~165 kDa apparent mass, use wet/tank transfer rather than semi-dry for more efficient transfer of this large protein. Use a low-percentage (6-8%) resolving gel, reduce methanol in transfer buffer to improve pore size, and extend transfer time (60-90 min, or overnight at low voltage/4°C) to ensure complete elution from the gel.
How should I block for a phosphorylation-sensitive CLASP2 antibody?
Because CLASP2 carries 28 UniProt-annotated modified residues, use 5% BSA rather than milk when the antibody targets a phosphorylated epitope, since milk contains phosphoproteins that can raise background or compete with phospho-specific binding. Use non-fat milk only if the antibody targets a non-phospho epitope.
How should I normalize CLASP2 signal for quantitation?
Given CLASP2's large size (~165 kDa observed) and cytoskeletal/centrosomal localization, avoid low-molecular-weight housekeeping proteins like GAPDH as loading controls, since they resolve far from CLASP2 and transfer with different efficiency. Use a total protein stain (e.g., Ponceau or stain-free gel) on the same blot for accurate normalization.
Why might I see multiple CLASP2 bands on a blot?
Multiple bands can reflect the three annotated isoforms, phosphorylation-driven mobility shifts across 28 modified residues, or partial proteolytic degradation of this large, repeat-containing protein during lysis. Use fresh lysates with protease and phosphatase inhibitors, and compare the band pattern to isoform sequence length to distinguish real isoforms from degradation products.
Boster reagents
Best CLASP2 Western Blot Antibodies
BosterBio's CLASP2 antibodies are among the best-performing WB antibodies on the market — well cited, thoroughly validated, and orthogonally cross-validated against negative tissues and complementary methods.
Real WB data
Anti-CLASP2 Antibody Picoband®
Cat # A03579-1
For CLASP2 Western blotting, the recommended Boster antibody below represents our best-performing option: extensively cited in the literature, rigorously validated through multiple independent experiments, and orthogonally cross-validated against negative tissue controls and complementary detection methods to ensure specificity and reproducible results.
Which to pick: Only one CLASP2 antibody is catalogued here, A03579-1, so it's the clear choice. It includes a genuine Western blot validation image (SDS-PAGE, 5-20% gel), giving direct evidence of specific band detection for your experiment.
Source: BosterBio CLASP2 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry O75122.
- Human Protein Atlas. CLASP2 tissue expression.