This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
CNN1 / Calponin-1 · Western blot design guide
Design a Western Blot for CNN1
Real validated CNN1 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-CNN1 WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Evidence assembled September 2026 · For research use; verify linked source records and product datasheet before use
CNN1 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 | ~33.2 kDa | |
| Observed band | 33 kDa | |
| Gel | 12–15% (standard starting point) | |
| Positive control | Endometrium (IHC candidate; verify WB) +2 more | |
| Negative control | Adipose tissue (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated CNN1 Western Blot Protocols
The PA2224 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | HELA at 40ug, JURKAT at 40ug, MCF-7 at 40ug (catalog PA2224) |
| Gel % | 12–15% (standard starting point) |
| Load | 20–30 µg total protein per lane; optimize for abundance (standard starting point) |
| Transfer | Standard semi-dry transfer; verify efficiency (standard starting point) |
| Membrane | 0.45 µm PVDF (standard starting point) |
| Blocking | 5% milk or 5% BSA in TBST (standard starting point) |
| Primary antibody | PA2224; use the WB datasheet starting dilution (standard starting point) |
| Primary incubation | Overnight at 4 °C (standard starting point) |
| Secondary antibody | Species-matched HRP conjugate at validated dilution (standard starting point) |
| Secondary incubation | 1 h at room temperature (standard starting point) |
| Wash | 3 × 5 min in TBST (standard starting point) |
| Detection | ECL; bracket exposures to avoid saturation (standard starting point) |
Section 2
What Is the Expected CNN1 Western Blot Band Size?
CNN1 is predicted at 33.2 kDa and observed at 33 kDa; the cause of the small difference is not established.
| Band at 33 kDa | Matches the empirical CNN1 band |
| Single band near 33 kDa | Consistent with the 33.2 kDa prediction; isoforms 1 and 2 need not resolve separately |
| Additional band at a different mass | Could reflect isoform 1 or 2, but band identity requires confirmation |
| Band with altered mobility | Could reflect ROCK2 phosphorylation; a visible shift is not established |
💡Expected CNN1 appearanceCNN1 is predicted at 33.2 kDa and observed at 33 kDa; confirm band identity with appropriate antibody and sample controls.
| Predicted molecular mass | 33.2 kDa predicted; the empirical band is 33 kDa |
| Full-length 297-residue sequence | Provides the basis for the 33.2 kDa prediction |
| Splice isoform 1 | May migrate differently from isoform 2; its mass is not supplied |
| Splice isoform 2 | May migrate differently from isoform 1; its mass is not supplied |
| Situation | Likely cause | Next action |
|---|---|---|
| Band higher than expected | Isoform identity or another cause is unconfirmed | Compare with the 33 kDa reference band and verify antibody specificity |
| Band lower than expected | Isoform identity or another cause is unconfirmed | Check band identity with a second CNN1 antibody |
| Multiple bands | Isoforms 1 and 2 are annotated, but distinct bands are not demonstrated | Check antibody specificity and compare samples with different CNN1 expression |
| Weak or no signal | CNN1 abundance or assay sensitivity may be insufficient | Include a known CNN1-positive sample and check loading and antibody performance |
| Fragments below expected size | Fragment identity is not established by the supplied features | Check sample integrity and confirm the bands with a second CNN1 antibody |
Experiment evidence
Western blot images showing CNN1 band
PA2224 · CNN1
M08065-1 · CNN1
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for CNN1 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for CNN1 in Western blot, you can use endometrium lysate, which has high HPA expression.
Positive control: Endometrium (IHC candidate; verify WB)
Negative control: Adipose tissue (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: HPA reports high expression in endometrium and no detection in adipose tissue, supporting a tissue-based positive and negative pair.
HPA tissue expression evidence for CNN1
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 |
|---|---|---|---|---|
| Endometrium | smooth muscle cells | High | Protein (IHC) | HPA → |
| Smooth muscle | smooth muscle cells | High | Protein (IHC) | HPA → |
| Breast | myoepithelial cells | Medium | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | adipocytes | Not detected | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | Not detected | Protein (IHC) | HPA → |
| Appendix | endocrine cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CNN1 Western Blot Tips
Deeper troubleshooting and optimisation questions for CNN1, answered from its protein features.
How should CNN1 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 CNN1 isoforms produce different bands?
Isoforms · UniProt lists isoforms 1 and 2. In isoform 2, residues 1–21 of the canonical sequence are replaced by a single methionine. This sequence difference could affect migration, but the features do not establish where either isoform runs. Confirm isoform identity before assigning a band.
Use an antibody that recognizes sequence shared by isoforms 1 and 2. If both produce detectable bands, define in advance whether to measure each band or their combined signal. The supplied features do not establish that the isoforms resolve separately.
Use an antibody that recognizes sequence shared by isoforms 1 and 2. If both produce detectable bands, define in advance whether to measure each band or their combined signal. The supplied features do not establish that the isoforms resolve separately.
Which CNN1 phosphorylation sites should I consider?
PTM · UniProt lists ROCK2 phosphorylation at Thr170, Ser175, Thr180, Thr184, and Thr259. These are UniProt coordinates; antibody or paper numbering may differ. Check which residue an antibody recognizes before interpreting a phosphorylation signal.
No. ROCK2 modifies five listed residues, but site annotation alone does not show that phosphorylation creates a visible doublet. Test phosphorylation directly before assigning either band; also consider the two annotated isoforms.
UniProt lists no glycosylation sites for CNN1. Its features therefore provide no basis to assign an unexpected band to glycosylation. Check band identity and the annotated isoform difference before assigning a cause.
No. ROCK2 modifies five listed residues, but site annotation alone does not show that phosphorylation creates a visible doublet. Test phosphorylation directly before assigning either band; also consider the two annotated isoforms.
UniProt lists no glycosylation sites for CNN1. Its features therefore provide no basis to assign an unexpected band to glycosylation. Check band identity and the annotated isoform difference before assigning a cause.
Does this guide establish induction of CNN1?
Induction · No general induction response is established by this guide. A pathway or gene-set association is not evidence of induction in a particular specimen. Verify the relevant treatment and control in a target-specific experiment.
How should transfer be checked for CNN1?
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 PA2224 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 CNN1 be quantified?
Quantitation · Standard workflow guidance: quantify only a validated target band within the linear exposure range. Use consistent sample preparation and loading, retain biological replicates, and avoid interpreting saturation or loading differences as regulation.
Does CNN1 show a band shift from its predicted mass?
Interpretation · The observed band is 33 kDa, close to the predicted 33.2 kDa. These values do not establish a meaningful shift. The listed phosphorylation sites alone do not demonstrate a visible change in migration.
CNN1 is listed as part of a cGMP kinase signaling complex with ACTA2, PLN, PRKG1, and ITPR1. Membership does not establish that the complex remains intact during Western blotting or produces a higher-mass band. Confirm the band’s identity independently.
CNN1 is listed as part of a cGMP kinase signaling complex with ACTA2, PLN, PRKG1, and ITPR1. Membership does not establish that the complex remains intact during Western blotting or produces a higher-mass band. Confirm the band’s identity independently.
Boster reagents
CNN1 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-Calponin/CNN1 Antibody Picoband®
Cat # PA2224
Real WB data
Anti-Calponin CNN1 Rabbit Monoclonal Antibody
Cat # M08065-1
PA2224 and M08065-1 are anti-CNN1 antibodies listed as reactive with human, mouse, and rat. Both have Western blot images: PA2224 shows a 33 kDa band in human cell lysates; M08065-1 shows an approximately 35 kDa band in human cells and mouse stomach. No rat sample is shown.
Which to pick: For mouse stomach lysate, M08065-1 has a matching WB image. For human cell lysates, both have images: PA2224 shows HeLa, Jurkat, and MCF-7; M08065-1 shows MCF-7, K562, U20S, and Caco-2. Neither supplied image shows a rat sample.
Source: BosterBio CNN1 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P51911.
- Human Protein Atlas. CNN1 tissue expression.
- PMC11176519 — target-verified WB comparison
- PMC10866439 — target-verified WB comparison
- PMC10805486 — target-verified WB comparison