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- Table of Contents
SMAD1 / SMAD family member 1 · Western blot design guide
Design a Western Blot for SMAD1
Source-linked SMAD1 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-SMAD1 WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Evidence assembled October 2026 · For research use; verify linked source records and product datasheet before use
SMAD1 Western Blot Experimental Design Guide
Expected bands, source-linked protocol options, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before running
| Expected band | ~52.3 kDa | |
| Observed band | 52 kDa | |
| Gel | 10% (catalog M00728-1) | |
| Positive control | Epididymis (IHC candidate; verify WB) +4 more | |
| Negative control | Adrenal gland (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated + Acetylated | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 2 isoform(s) |
Section 1
Source-Linked SMAD1 Western Blot Protocol Options
The M00728-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human Hela, human Jurkat, human SIHA, human HepG2 (catalog M00728-1) |
| Gel % | 10% (catalog M00728-1) |
| Load | 30 ug; reducing conditions (catalog M00728-1) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog M00728-1) |
| Membrane | nitrocellulose membrane (catalog M00728-1) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog M00728-1) |
| Primary antibody | M00728-1 · 1:500 (catalog M00728-1) |
| Primary incubation | overnight at 4°C (catalog M00728-1) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog M00728-1) |
| Secondary incubation | 1.5 hour at RT (catalog M00728-1) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog M00728-1) |
| Detection | ECL (catalog M00728-1) |
Section 2
What Is the Expected SMAD1 Western Blot Band Size?
SMAD1 is predicted at 52.3 kDa and observed near 52 kDa; the small difference has no established cause in the supplied evidence.
| Band near 52 kDa | Matches the observed SMAD1 band and its 52.3 kDa predicted mass; confirm identity with a specificity control |
| Closely spaced doublet near 52 kDa | Could reflect different phosphorylation states; a visible shift has not been established |
| Multiple bands at different positions | Could reflect isoforms 1 and 2, although their relative masses and separation are unknown |
| Weak band in a cytoplasmic fraction | SMAD1 can move to the nucleus when complexed with SMAD4 |
💡Expected SMAD1 appearanceSMAD1 has a predicted mass of 52.3 kDa and an observed band near 52 kDa in reducing whole-cell lysates; confirm band identity with an appropriate specificity control.
| Predicted SMAD1 mass | 52.3 kDa predicts a band near the observed 52 kDa |
| Isoform 1 | May differ in size from isoform 2, but its mass and migration are not supplied |
| Isoform 2 | May differ in size from isoform 1, but its mass and migration are not supplied |
| Phosphorylation at Thr322, Ser463 and Ser465 | May affect migration, but no visible size shift is established |
| Situation | Likely cause | Next action |
|---|---|---|
| Band higher than expected | A phosphorylation state or another band identity is possible; its effect on migration is unproven | Compare phosphatase-treated and untreated samples and verify the band with SMAD1 depletion |
| Band lower than expected | An alternate isoform or proteolysis is possible; isoform masses are unknown | Check sample integrity and test whether SMAD1 depletion removes the band |
| Multiple bands | Isoforms 1 and 2 or different phosphorylation states are possible | Use SMAD1 depletion and a phosphatase comparison to identify responsive bands |
| Weak or no signal | SMAD1 may be enriched in the nucleus rather than the sampled cytoplasmic fraction | Check both nuclear and cytoplasmic fractions with fraction markers |
| Fragments below expected size | Sample proteolysis is possible; no SMAD1 cleavage product is specified | Prepare fresh lysate with protease inhibitors and confirm fragment identity by SMAD1 depletion |
Experiment evidence
Western blot images showing SMAD1 band
M00728-1 · SMAD1
M00728 · SMAD1
PB9395 · SMAD1
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for SMAD1 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for SMAD1 in Western blot, you can use epididymis tissue, which shows high HPA expression.
Positive control: Epididymis (IHC candidate; verify WB)
Negative control: Adrenal gland (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside the samples.
⚠️Feasibility: HPA identifies both high-expression and not-detected tissues, making tissue controls feasible for this intracellular protein.
HPA tissue expression evidence for SMAD1
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.
Higher expression tissues · candidate positive controls from IHC
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Epididymis | glandular cells | High | Protein (IHC) | HPA → |
| Stomach | glandular cells | High | Protein (IHC) | HPA → |
| Appendix | glandular cells | Medium | Protein (IHC) | HPA → |
| Breast | glandular cells | Medium | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | Medium | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adrenal gland | glandular cells | Not detected | Protein (IHC) | HPA → |
| Esophagus | squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Liver | cholangiocytes | Not detected | Protein (IHC) | HPA → |
| Ovary | ovarian stroma cells | Not detected | Protein (IHC) | HPA → |
| Parathyroid gland | glandular cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced SMAD1 Western Blot Tips
Deeper troubleshooting and optimisation questions for SMAD1, answered from its protein features.
How should SMAD1 band migration be interpreted?
Band shift · Use the separately labelled calculated mass and catalog-observed evidence above. A sequence annotation does not establish an observed migration shift. Verify target identity with orthogonal controls.
Could SMAD1 isoforms produce different bands?
Isoforms · UniProt lists isoforms 1 and 2. Relative to canonical isoform 1, isoform 2 lacks residues 13–133 and 259–465 and has substitutions at 1–12 and 220–258. It could therefore migrate differently. Check whether the antibody epitope is retained before assigning a band to either isoform.
Which SMAD1 modifications matter when interpreting a phospho-band?
PTM · Canonical UniProt coordinates list Thr322 phosphorylation by MINK1, TNIK and MAP4K4, plus phosphorylation at Ser463 and Ser465. Met1 is N-acetylated. Isoform 2 lacks the canonical Thr322, Ser463 and Ser465 regions. Check the antibody's target site and numbering convention; modification sites alone do not establish a visible band shift.
How might ligand stimulation change the SMAD1 signal?
Induction · UniProt describes SMAD1 as cytoplasmic without ligand and moving to the nucleus when complexed with SMAD4. It also describes trimer formation with SMAD1 and SMAD4 after C-terminal phosphorylation. For a ligand response, compare nuclear and cytoplasmic fractions and measure the relevant phospho-signal alongside total SMAD1.
How should transfer be checked for SMAD1?
Transfer · Standard workflow guidance: verify transfer efficiency for the intended target size before interpreting a weak signal. Use total-protein assessment and optimize transfer for the membrane, gel and apparatus; the labelled catalog values take precedence.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the M00728-1 datasheet where specified. Otherwise compare 5% milk or 5% BSA in TBST; for a phospho-specific assay start with BSA. Optimize background and specific signal with matched controls.
How should SMAD1 be quantified across cellular fractions?
Quantitation · Measure nuclear and cytoplasmic fractions separately if localization is the endpoint: UniProt places SMAD1 in both compartments and describes ligand-dependent nuclear migration with SMAD4. For phosphorylation, report the site-specific phospho-signal alongside total SMAD1 in the same samples.
Why is the SMAD1 band near 52 kDa?
Interpretation · The supplied observed band is 52 kDa, close to the 52.3 kDa predicted mass. UniProt lists modifications, but their presence alone does not establish a visible shift or explain any mass difference.
First check antibody epitope coverage against isoform 2, which lacks canonical residues 13–133 and 259–465. Then check whether the antibody recognizes total SMAD1 or a specified phosphosite. UniProt lists phosphorylation and N-terminal acetylation, but those features alone cannot identify an unexpected band or prove a migration shift.
First check antibody epitope coverage against isoform 2, which lacks canonical residues 13–133 and 259–465. Then check whether the antibody recognizes total SMAD1 or a specified phosphosite. UniProt lists phosphorylation and N-terminal acetylation, but those features alone cannot identify an unexpected band or prove a migration shift.
Boster reagents
SMAD1 Western Blot Antibodies
Catalog antibodies with Western blot application and product-specific WB images. Evaluate suitability with the reported sample, controls and experimental conditions.
Real WB data
Anti-Smad1 Rabbit Monoclonal Antibody
Cat # M00728-1
Real WB data
Anti-Smad1 Rabbit Monoclonal Antibody
Cat # M00728
Real WB data
Anti-SMAD1 Antibody Picoband®
Cat # PB9395
Three listed anti-SMAD1 antibodies have Western blot images. M00728-1 and PB9395 report bands at approximately 52 kDa in the samples described; M00728 shows 293T lysate. The supplied evidence is limited to these product images and captions.
Which to pick: For human cell lysates, M00728-1 has documented HeLa, Jurkat, SIHA, and HepG2 lanes; M00728 shows 293T lysate. PB9395 shows human cell lysates plus mouse and rat cardiac and skeletal muscle lysates. Choose based on your sample and listed reactivity.
Source: BosterBio SMAD1 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q15797.
- Human Protein Atlas. SMAD1 tissue expression.
- PMC10972565 — target-verified WB comparison
- PMC10723262 — target-verified WB comparison
- PMC5652667 — target-verified WB comparison