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- Table of Contents
SMAD4 · Western blot design guide
SMAD4 Western Blot Planning Guide
Plan a SMAD4 Western blot around the catalog-observed 60.4 kDa band, image-backed PB9397 evidence, HPA controls, and verified protocol records.
Evidence assembled July 2026 · For research use; verify linked source records and product datasheet before use
SMAD4 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 | 60.4 kDa | |
| Observed band | Not reported — verify product WB image | |
| Gel | 12-15% | |
| Positive control | Colon | |
| Negative control | Lymph node |
Important caveats
Reasons your observed band may differ from the expected size.
| ⓘ | Calculated mass | 60.4 kDa |
| ⓘ | Localization | Cytoplasm / Nucleus |
| ⓘ | Processing / PTM | Record-dependent |
| ⓘ | Reactivity | Human / Mouse / Rat |
Section 1
Real Curated SMAD4 Western Blot Protocols
Start with the molecular-weight rule, then compare verified publication-derived conditions.
| Sample / lysate | Appendix |
| 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 | PB9397 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 SMAD4 Western Blot Band Size?
Use the product-observed 60.4 kDa band as the primary planning value and retain the UniProt calculated mass as context.
| 60.4 kDa | Matches the authoritative product WB observation. |
| 60.4 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 SMAD4 appearancePlan around 60.4 kDa and keep the calculated mass as supporting context.
| Catalog-observed band | 60.4 kDa; use this as the primary experimental expectation. |
| Calculated mass | 60.4 kDa from UniProt Q13485; 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 PB9397. |
| Situation | Likely cause | Next action |
|---|---|---|
| 60.4 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 SMAD4 band
PB9397 · SMAD4
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for SMAD4 Western blot
🧪Use Colon as the first positive-control candidate and Lymph node as the HPA Not detected negative candidate.
Positive control: Colon (High)
Negative control: Lymph node (Not detected)
HPA protein score determines control status; other expression data is supporting context only.
HPA tissue expression evidence for SMAD4
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 |
|---|---|---|---|---|
| Colon | Reported tissue cells | High | Protein (HPA) | HPA → |
| Lung | Reported tissue cells | High | Protein (HPA) | HPA → |
| Appendix | Reported tissue cells | High | Protein (HPA) | HPA → |
| Duodenum | Reported tissue cells | High | Protein (HPA) | HPA → |
Section 3
Advanced SMAD4 Western Blot Tips
Deeper troubleshooting and optimisation questions for SMAD4, answered from its protein features.
Which band should guide the blot?
Use 60.4 kDa, the observation attached to the authoritative PB9397 WB record.
How should calculated mass be interpreted?
Treat the UniProt calculated mass as context; it does not replace the catalog-observed 60.4 kDa expectation.
Which positive control should I start with?
Start with Colon, the lead HPA protein-expression candidate.
Which negative control is defensible?
Use Lymph node 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 SMAD4 Western blot?
Use the transfer method in the recommended protocol and verify transfer before blocking.
How should PB9397 be started?
Start at the linked datasheet condition and run a three-point primary-antibody dilution test.
Which publication-derived protocols can I compare?
No verified publication protocol was supplied; use only the deterministic recommended protocol.
Boster reagents
SMAD4 Western Blot Reagents
Human/Mouse/Rat-reactive SMAD4 Western blot reagents with authoritative product imagery.
Real WB data
Anti-Smad4 Antibody Picoband®
Cat # PB9397
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 Q13485
- Human Protein Atlas — SMAD4
- PB9397 product record
- PMC6345154 — SMAD4 Gene Mutation Renders Pancreatic Cancer Resistance to Radiotherapy through Promotion of Autophagy (Clinical cancer research : an official journal of the American Association for Cancer Research, 2018)
- PMC5354968 — Aberrant Phosphorylation of SMAD4 Thr277-Mediated USP9x-SMAD4 Interaction by Free Fatty Acids Promotes Breast Cancer Metastasis (Cancer research, 2017)
- PMC10243371 — Deubiquitinase USP13 promotes extracellular matrix expression by stabilizing Smad4 in lung fibroblast cells (Translational research : the journal of laboratory and clinical medicine, 2020)