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- Table of Contents
CAST · Western blot design guide
CAST Western Blot Planning Guide
Plan a CAST Western blot around the catalog-observed 55 kDa band, image-backed A00337 evidence, HPA controls, and verified protocol records.
Evidence assembled July 2026 · For research use; verify linked source records and product datasheet before use
CAST Western Blot Experimental Design Guide
Expected bands, documented protocol parameters, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before running
| Expected band | 55 kDa | |
| Observed band | Not reported — verify product WB image | |
| Gel | 12-15% | |
| Positive control | Bronchus | |
| Negative control | Hippocampus |
Important caveats
Reasons your observed band may differ from the expected size.
| ⓘ | Calculated mass | 55 kDa |
| ⓘ | Localization | Nucleus, nucleolus / Chromosome |
| ⓘ | Processing / PTM | Record-dependent |
| ⓘ | Reactivity | Human |
Section 1
Real Curated CAST Western Blot Protocols
Start with the molecular-weight rule, then compare verified publication-derived conditions.
| Sample / lysate | Bronchus |
| Gel % | 12-15% |
| Load | 20-30 µg total protein per lane |
| Transfer | Semi-dry, standard transfer |
| Membrane | 0.45 µm PVDF |
| Blocking | 5% non-fat milk or 5% BSA in TBST |
| Primary | A00337 at datasheet starting dilution |
| Primary incubation | Overnight at 4 °C with gentle agitation |
| Secondary | Species-matched HRP conjugate at validated dilution |
| Wash | 3 × 5 min in TBST |
| Detection | Chemiluminescent substrate |
| Exposure | Bracket exposures to avoid saturation |
Section 2
What Is the Expected CAST Western Blot Band Size?
Use the product-observed 55 kDa band as the primary planning value and retain the UniProt calculated mass as context.
| 55 kDa | Matches the authoritative product WB observation. |
| 55 kDa calculated | Use as UniProt context, not as a replacement observed band. |
| Unexpected additional signal | Do not assign identity without orthogonal positive/negative controls. |
💡Expected CAST appearancePlan around 55 kDa and keep the calculated mass as supporting context.
| Catalog-observed band | 55 kDa; use this as the primary experimental expectation. |
| Calculated mass | 55 kDa from UniProt O15446; retain as context. |
| Gel selection | 12-15%; shared with the recommended protocol and poster. |
| Specificity check | Compare the lead HPA positive and negative controls with A00337. |
| Situation | Likely cause | Next action |
|---|---|---|
| 55 kDa | Matches the authoritative product WB observation. | Confirm with orthogonal controls and the linked product record. |
| Additional band | May reflect processing, modification, or non-specific signal. | Run a dilution series and compare positive/negative controls. |
| Weak signal | Target abundance or transfer may be limiting. | Verify transfer, increase positive-control abundance, and bracket exposure. |
Experiment evidence
Western blot images showing CAST band
A00337 · CAST
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for CAST Western blot
🧪Use Bronchus as the first positive-control candidate and Hippocampus as the HPA Not detected negative candidate.
Positive control: Bronchus (High)
Negative control: Hippocampus (Not detected)
HPA protein score determines control status; other expression data is supporting context only.
HPA tissue expression evidence for CAST
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 |
|---|---|---|---|---|
| Bronchus | Reported tissue cells | High | Protein (HPA) | HPA → |
| Nasopharynx | Reported tissue cells | High | Protein (HPA) | HPA → |
| Breast | Reported tissue cells | Medium | Protein (HPA) | HPA → |
| Adrenal gland | Reported tissue cells | Medium | Protein (HPA) | HPA → |
Section 3
Advanced CAST Western Blot Tips
Deeper troubleshooting and optimisation questions for CAST, answered from its protein features.
Which band should guide the blot?
Use 55 kDa, the observation attached to the authoritative A00337 WB record.
How should calculated mass be interpreted?
Treat the UniProt calculated mass as context; it does not replace the catalog-observed 55 kDa expectation.
Which positive control should I start with?
Start with Bronchus, the lead HPA protein-expression candidate.
Which negative control is defensible?
Use Hippocampus as an orthogonal HPA Not detected candidate.
Which gel should I use?
Use 12-15% consistently across the quick facts, protocol table, and poster.
What transfer method to use for CAST Western blot?
Use the transfer method in the recommended protocol and verify transfer before blocking.
How should A00337 be started?
Start at the linked datasheet condition and run a three-point primary-antibody dilution test.
Which publication-derived protocols can I compare?
Compare only the supplied target-verified records: PMC3715600, PMC6848861.
Boster reagents
CAST Western Blot Reagents
Human-reactive CAST Western blot reagents with authoritative product imagery.
Real WB data
Anti-Calpastatin/CAST Antibody Picoband®
Cat # A00337
Only image-backed, WB-validated Human/Mouse/Rat recommendations from the prepared catalog evidence are shown.
Source: prepared picoband-wb product evidence; each card retains its own SKU, URL, observed band, and authoritative WB image.
References
- UniProt O15446
- Human Protein Atlas — CAST
- A00337 product record
- PMC3715600 — Modulation by Syk of Bcl-2, calcium and the calpain-calpastatin proteolytic system in human breast cancer cells.
- PMC6848861 — Calpastatin participates in the regulation of cell migration in BAP1-deficient uveal melanoma cells.