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- Table of Contents
CHAD / Chondroadherin · Western blot design guide
Design a Western Blot for CHAD
Real validated CHAD Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-CHAD 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
CHAD 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 | ~40.5 kDa | |
| Gel | 12–15% (standard starting point) | |
| Positive control | Adrenal gland (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 | Glycosylated + Cleaved | |
| Caveat | Glycosylation-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated CHAD Western Blot Protocols
The A03177 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | 3T3 cell lysate (catalog A03177) |
| 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 | A03177 · 1 μg/ml (catalog A03177) |
| 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 CHAD Western Blot Band Size?
CHAD has a predicted 40.5 kDa precursor; signal-peptide cleavage and Ser144 O-linked GalNAc may affect migration, but no empirical band size or shift is established.
| Band near 40.5 kDa | consistent with the predicted precursor size; identity requires confirmation |
| Band below 40.5 kDa | could reflect removal of the signal peptide at residues 1–22 |
| Little or no band in whole-cell lysate | CHAD is secreted into the extracellular matrix |
| Band with altered migration | O-linked GalNAc at Ser144 could contribute, but a visible shift is unproven |
💡Expected CHAD appearanceUniProt predicts a 40.5 kDa precursor; signal-peptide cleavage and O-linked GalNAc at Ser144 may affect migration, but no empirical band size is supplied, so confirm identity with appropriate controls.
| Predicted 40.5 kDa mass | sets the reference size for the precursor |
| 359-residue precursor | underlies the predicted mass; mature size is not supplied |
| Signal peptide at residues 1–22 | cleavage could yield a smaller mature protein |
| O-linked GalNAc at Ser144 | may affect apparent size; no visible shift is established |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | CHAD is secreted into the extracellular matrix | check conditioned medium or extracellular matrix fractions |
| Band higher than expected | Ser144 O-linked GalNAc may affect migration, but the cause is unproven | compare treated and untreated samples and confirm identity with a second antibody |
| Band lower than expected | the signal peptide may have been removed | compare cell and extracellular samples and confirm identity with a second antibody |
| Multiple bands | precursor and mature forms or nonspecific binding may be present | compare fractions and verify bands with a second antibody |
| Weak or no signal | extracellular CHAD may be scarce in the sampled fraction | enrich conditioned medium or extracellular matrix material |
| Fragments below expected size | sample degradation is possible | prepare samples with protease inhibitors and verify fragment identity |
Experiment evidence
Western blot images showing CHAD band
A03177 · CHAD
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for CHAD Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for CHAD in Western blot, you can use adrenal gland tissue.
Positive control: Adrenal gland (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: CHAD is secreted into extracellular matrix, so whole-cell lysates may show weak signal.
HPA tissue expression evidence for CHAD
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 |
|---|---|---|---|---|
| Adrenal gland | glandular cells | Medium | Protein (IHC) | HPA → |
| Appendix | enterocytes - Microvilli | Medium | Protein (IHC) | HPA → |
| Cervix | glandular cells | Medium | Protein (IHC) | HPA → |
| Colon | enterocytes - Microvilli | Medium | Protein (IHC) | HPA → |
| Duodenum | endocrine 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 → |
| Bone marrow | hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | adipocytes | Not detected | Protein (IHC) | HPA → |
| Caudate | glial cells | Not detected | Protein (IHC) | HPA → |
| Cerebellum | cells in granular layer | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CHAD Western Blot Tips
Deeper troubleshooting and optimisation questions for CHAD, answered from its protein features.
How should CHAD 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 CHAD isoforms explain multiple bands?
Isoforms · The supplied UniProt features list one isoform and no alternative sequence. They do not support assigning multiple bands to annotated CHAD isoforms.
Which CHAD modification site matters when interpreting a band?
PTM · UniProt lists an O-linked GalNAc-type glycosylation site at Ser144, numbered on the full-length sequence. Check the numbering convention before comparing it with an antibody or paper. The site’s presence does not establish glycan occupancy or a visible band shift.
Does this guide establish induction of CHAD?
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 CHAD?
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 A03177 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.
Which sample fraction is relevant for CHAD quantitation?
Quantitation · CHAD is annotated in the extracellular space and extracellular matrix. Choose a fraction that captures the CHAD pool of interest, and use the same fraction and preparation across samples. A signal from one fraction alone may not represent total CHAD.
Why might CHAD migrate differently from its predicted 40.5 kDa?
Interpretation · The 40.5 kDa prediction reflects the 359-residue sequence. CHAD has a signal peptide at residues 1–22 and an O-linked glycosylation site at Ser144 (UniProt full-length numbering). Processing or glycosylation could affect migration, but these features alone cannot predict a band position or explain a measured difference. No observed band size was supplied.
CHAD has a signal peptide at residues 1–22 and is annotated as secreted. A processed protein may therefore differ in mass from the full-length 40.5 kDa prediction. The supplied features do not give an observed band position.
UniProt lists three disulfide bonds. Compare reducing and nonreducing lanes if migration is unexpected, and interpret any difference cautiously: the features do not predict its size or direction.
CHAD is mostly monomeric by similarity and interacts with collagen type II. Its three disulfide bonds and glycosylation site warrant checking preparation and reducing conditions. These annotations do not establish that a higher band is a CHAD complex or a glycosylated form; confirm band identity experimentally.
CHAD has a signal peptide at residues 1–22 and is annotated as secreted. A processed protein may therefore differ in mass from the full-length 40.5 kDa prediction. The supplied features do not give an observed band position.
UniProt lists three disulfide bonds. Compare reducing and nonreducing lanes if migration is unexpected, and interpret any difference cautiously: the features do not predict its size or direction.
CHAD is mostly monomeric by similarity and interacts with collagen type II. Its three disulfide bonds and glycosylation site warrant checking preparation and reducing conditions. These annotations do not establish that a higher band is a CHAD complex or a glycosylated form; confirm band identity experimentally.
Boster reagents
CHAD 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-Chondroadherin CHAD Antibody
Cat # A03177
A03177 is an anti-CHAD antibody listed as reactive with human, mouse, and rat. Its WB image shows Chondroadherin in 3T3 cell lysate at 1 μg/ml; the supplied evidence does not show WB results in other samples.
Which to pick: A03177 is the only listed CHAD antibody and has a WB image using 3T3 cell lysate. Check its listed reactivity against your sample species, and optimize conditions for your own lysate.
Source: BosterBio CHAD gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry O15335.
- Human Protein Atlas. CHAD tissue expression.
- PMC4573979 — target-verified WB comparison
- PMC5601137 — target-verified WB comparison
- PMC3670915 — target-verified WB comparison