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- Table of Contents
CGN / Cingulin · Western blot design guide
Design a Western Blot for CGN
Real validated CGN Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-CGN 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
CGN 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 | ~137.1 kDa | |
| Gel | 8–10% (standard starting point) | |
| Positive control | Appendix (IHC candidate; verify WB) +4 more | |
| Negative control | Adipose tissue (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 C7 membership | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated CGN Western Blot Protocols
The A02373 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | extracts of various cell lines, (catalog A02373) |
| Gel % | 8–10% (standard starting point) |
| Load | 20–30 µg total protein per lane; optimize for abundance (standard starting point) |
| Transfer | Wet/tank transfer; optimize duration (standard starting point) |
| Membrane | 0.45 µm PVDF (standard starting point) |
| Blocking | Blocking buffer: 3% nonfat dry milk in TBST (catalog A02373) |
| Primary antibody | A02373 · 1:1000 (catalog A02373) |
| Primary incubation | Overnight at 4 °C (standard starting point) |
| Secondary antibody | Goat Anti-Rabbit IgG, 1:10000 (catalog A02373) |
| Secondary incubation | 1 h at room temperature (standard starting point) |
| Wash | 3 × 5 min in TBST (standard starting point) |
| Detection | ECL (catalog A02373) |
Section 2
What Is the Expected CGN Western Blot Band Size?
Cingulin is predicted at 137.1 kDa; homodimerization and isoforms could affect bands, but their migration effects have not been demonstrated here.
| Band near 137.1 kDa | Consistent with predicted cingulin size; confirm its identity with antibody controls |
| Band near 274 kDa | Could reflect the documented homodimer if it survives electrophoresis |
| Multiple bands at different positions | Could reflect isoforms 1 and 2; their migration is not established |
| Faint band in whole-cell lysate | Could reflect limited cingulin in the sampled tight-junction material |
💡Expected CGN appearanceCingulin has a predicted mass of 137.1 kDa, but no empirical band size is supplied; homodimerization and alternative splicing suggest possibilities, so confirm any band with ordinary identity controls.
| Predicted cingulin mass | 137.1 kDa is the sequence-based reference, not a measured band position |
| Homodimer formation | Could yield a band near twice the monomer mass if the dimer survives electrophoresis |
| Isoform 1 | Its individual mass and migration are not supplied |
| Isoform 2 | Its individual mass and migration are not supplied |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | The sample may contain little tight-junction cingulin | Compare with a junction-rich positive-control lysate |
| Band higher than expected | A homodimer may persist during electrophoresis | Compare fully denatured and standard preparations |
| Band lower than expected | An alternate isoform is possible, but its size is unknown | Check the antibody epitope and confirm the band with an independent antibody |
| Multiple bands | Isoforms 1 and 2 or phosphorylation may contribute, without established migration patterns | Compare with an independent antibody and a phosphatase-treated aliquot |
| Weak or no signal | Cingulin is localized at cell junctions | Check sample integrity and compare with a junction-rich positive control |
Experiment evidence
Western blot images showing CGN band
A02373 · CGN
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for CGN Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for CGN in Western blot, you can use appendix tissue lysate, which HPA scores High.
Positive control: Appendix (IHC candidate; verify WB)
Negative control: Adipose tissue (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside the samples.
⚠️Feasibility: CGN localizes to cell junctions, so signal may depend on the proportion of junction-bearing cells in the lysate.
HPA tissue expression evidence for CGN
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 |
|---|---|---|---|---|
| Appendix | enterocytes - Microvilli | High | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Colon | enterocytes - Microvilli | High | Protein (IHC) | HPA → |
| Duodenum | enterocytes - Microvilli | High | Protein (IHC) | HPA → |
| Fallopian tube | glandular cells | High | 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 → |
| Bone marrow | hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Cerebral cortex | endothelial cells | Not detected | Protein (IHC) | HPA → |
| Heart muscle | cardiomyocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CGN Western Blot Tips
Deeper troubleshooting and optimisation questions for CGN, answered from its protein features.
Where should the main CGN band appear?
Band shift · The predicted mass of canonical CGN is 137.1 kDa. No empirical apparent band position is supplied, so use 137.1 kDa as a reference, not an exact migration target. The listed modifications alone do not establish a visible shift.
Could CGN isoforms produce different bands?
Isoforms · UniProt lists isoforms 1 and 2. In isoform 2, canonical residues 704–777 are replaced and residues 778–1203 are missing. A shorter band could therefore be consistent with isoform 2, but band position alone cannot identify it.
Check the antibody epitope against the isoform sequences. Canonical residues 778–1203 are absent from isoform 2, while residues 704–777 are replaced. An epitope in either region may change which isoform the antibody detects.
No. Isoform 2 replaces canonical residues 704–777, which include phosphothreonine 712, and lacks residues 778–1203, which include phosphoserines 1175, 1176, and 1182. Do not assign those canonical sites to isoform 2 without checking its sequence and numbering.
Check the antibody epitope against the isoform sequences. Canonical residues 778–1203 are absent from isoform 2, while residues 704–777 are replaced. An epitope in either region may change which isoform the antibody detects.
No. Isoform 2 replaces canonical residues 704–777, which include phosphothreonine 712, and lacks residues 778–1203, which include phosphoserines 1175, 1176, and 1182. Do not assign those canonical sites to isoform 2 without checking its sequence and numbering.
Which CGN modifications matter when interpreting bands?
PTM · UniProt lists phosphoserine at 95, 96, 135, 137, 140, 155, 165, 214, 217, 258, 276, 338, 351, 1175, 1176, and 1182; N6-acetyllysine at 579; and phosphothreonine at 712. These are UniProt canonical coordinates and may differ from antibody or paper numbering. Their presence does not establish a visible band shift.
Does this guide establish induction of CGN?
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 CGN?
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 A02373 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 CGN bands be quantified across samples?
Quantitation · CGN localizes to cell junctions, including tight junctions. Keep sample preparation consistent across samples, and confirm that the quantified band is recognized by the antibody and corresponds to the intended isoform. If multiple bands appear, report which one was measured.
Could a high-mass CGN band represent a dimer?
Interpretation · UniProt describes CGN as a homodimer. A high-mass band may warrant checking whether it represents a dimer, but homodimerization alone does not establish that identity or predict its migration in a Western blot.
First compare it with isoform 2, which lacks canonical residues 778–1203, and check whether the antibody epitope is retained. The supplied features do not establish the identity of any observed band, so a smaller band cannot be assigned to an isoform from size alone.
First compare it with isoform 2, which lacks canonical residues 778–1203, and check whether the antibody epitope is retained. The supplied features do not establish the identity of any observed band, so a smaller band cannot be assigned to an isoform from size alone.
Boster reagents
CGN 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-Cingulin CGN Antibody
Cat # A02373
The listed anti-CGN antibody, A02373, is reported for human reactivity and has a Western blot image of extracts from various cell lines. The caption reports 25 µg per lane and a 1:1000 primary dilution; it does not identify the cell lines.
Which to pick: A02373 is the only listed CGN antibody. It has a Western blot image and reported human reactivity; consider it for human samples, while noting that the imaged cell lines are unnamed.
Source: BosterBio CGN gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q9P2M7.
- Human Protein Atlas. CGN tissue expression.
- PMC10855320 — target-verified WB comparison
- PMC10630877 — target-verified WB comparison
- PMC4976216 — target-verified WB comparison