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- Table of Contents
COL5A3 / Collagen alpha-3(V) chain · Western blot design guide
Design a Western Blot for COL5A3
Source-linked COL5A3 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-COL5A3 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
COL5A3 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 | ~172.1 kDa | |
| Gel | 8–10% (standard starting point) | |
| Positive control | Cervix (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 | Modification-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 1 isoform(s) |
Section 1
Source-Linked COL5A3 Western Blot Protocol Options
The A10525 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | various cells (catalog A10525) |
| 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 | 5% milk or 5% BSA in TBST (standard starting point) |
| Primary antibody | A10525; 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 COL5A3 Western Blot Band Size?
COL5A3 has a predicted 172.1 kDa precursor; cleavage and glycosylation could alter migration, but no empirical band size or feature-specific shift is established.
| Band near 172.1 kDa | Compatible with the predicted full-length chain; identity requires validation |
| Band slightly below the precursor position | May reflect cleavage of the 29-residue signal peptide |
| Band above the precursor position | May reflect modification at the listed glycosylation sites |
| Broad band or smear | May reflect variable modification at the chondroitin sulfate attachment site |
| Little or no band in whole-cell lysate | Consistent with secretion into the extracellular matrix |
💡Expected COL5A3 appearanceUniProt predicts a 172.1 kDa precursor; signal-peptide cleavage and glycosylation may affect migration, but no empirical band size is supplied, so confirm identity with appropriate controls.
| Predicted full-length mass | 172.1 kDa is the calculated precursor mass, not a measured band |
| Signal peptide at residues 1–29 | Cleavage produces a shorter mature chain; its apparent mass is not supplied |
| N-linked glycosylation at Asn102 | May increase apparent mass; a visible shift is not established |
| N-linked glycosylation at Asn141 | May increase apparent mass; a visible shift is not established |
| O-linked chondroitin sulfate at Ser349 | May increase or broaden apparent mass; the extent is not supplied |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | COL5A3 is secreted into the extracellular matrix | Check conditioned medium or an extracellular matrix preparation |
| Band higher than expected | Modification at the listed glycosylation sites is possible | Compare with a validated COL5A3 control and assess glycosylation |
| Band lower than expected | The signal peptide may have been cleaved | Compare precursor and mature-chain detection with a validated antibody control |
| Broad smear instead of sharp band | Variable chondroitin sulfate modification at Ser349 is possible | Assess glycosylation and confirm the signal with a COL5A3-specific control |
| Multiple bands | Precursor processing or variable glycosylation is possible | Check reducing conditions and verify each band with an independent antibody |
| Weak or no signal | Secreted COL5A3 may be scarce in the tested cellular fraction | Test conditioned medium or extracellular matrix alongside a positive control |
Experiment evidence
Western blot images showing COL5A3 band
A10525 · COL5A3
A10525 · COL5A3
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for COL5A3 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for COL5A3 in Western blot, you can use cervix tissue, which HPA rates High.
Positive control: Cervix (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: Because COL5A3 is secreted into the extracellular matrix, whole-cell lysates may give weak signal; consider matrix-enriched material.
HPA tissue expression evidence for COL5A3
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 |
|---|---|---|---|---|
| Cervix | squamous epithelial cells | High | Protein (IHC) | HPA → |
| Parathyroid gland | glandular cells | High | Protein (IHC) | HPA → |
| Small intestine | glandular cells | High | Protein (IHC) | HPA → |
| Testis | Leydig cells | High | Protein (IHC) | HPA → |
| Tonsil | squamous epithelial 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 → |
| Endometrium | cells in endometrial stroma | Not detected | Protein (IHC) | HPA → |
| Epididymis | glandular cells | Not detected | Protein (IHC) | HPA → |
| Hippocampus | glial cells | Not detected | Protein (IHC) | HPA → |
| Lung | alveolar cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced COL5A3 Western Blot Tips
Deeper troubleshooting and optimisation questions for COL5A3, answered from its protein features.
How should COL5A3 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 a second band represent a COL5A3 isoform?
Isoforms · The supplied features list one isoform and no alternative sequence. Do not assign a second band to a documented COL5A3 isoform on this evidence alone; assess it against the listed processing and modification features.
Which COL5A3 glycosylation sites matter when interpreting a band?
PTM · The supplied UniProt features list N-linked sites at Asn102 and Asn141 and an O-linked chondroitin sulfate site at Ser349. These are UniProt canonical-sequence coordinates; check the numbering used by your antibody or paper before comparing sites. Their presence alone does not demonstrate a visible shift.
Does this guide establish induction of COL5A3?
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.
What transfer method to use for COL5A3 Western blot?
Transfer · COL5A3 is a 1,745-residue collagen chain with a predicted mass of 172.1 kDa. The supplied features do not specify a transfer method. Check transfer efficiency for protein in this size range before interpreting a weak or absent signal.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A10525 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 COL5A3 be quantified across tissue samples?
Quantitation · COL5A3 is secreted into the extracellular matrix. The supplied oligomer description places alpha-3(V) in placental trimers, while most tissues have alpha-1(V)/alpha-2(V) trimers. Account for tissue and extracellular-matrix sampling when comparing COL5A3 signals; a difference in band intensity alone does not establish altered expression.
Why might COL5A3 migrate differently from its predicted 172.1 kDa mass?
Interpretation · The 172.1 kDa prediction does not account for every possible effect of signal peptide removal, glycosylation, or collagen structure on apparent migration. No observed band size is supplied, so these features cannot establish the size or cause of a shift.
COL5A3 has a signal peptide at residues 1–29 and is secreted into the extracellular matrix. Consider whether the sample contains intracellular precursor or secreted protein when comparing bands. The feature does not establish that both forms will be visible.
Check sample source and processing against COL5A3's secreted location, 1–29 signal peptide, three listed glycosylation sites, and five disulfide bonds. Compare bands under the sample preparation conditions used. These features suggest possibilities to test but do not identify an unexpected band by themselves.
COL5A3 has a signal peptide at residues 1–29 and is secreted into the extracellular matrix. Consider whether the sample contains intracellular precursor or secreted protein when comparing bands. The feature does not establish that both forms will be visible.
Check sample source and processing against COL5A3's secreted location, 1–29 signal peptide, three listed glycosylation sites, and five disulfide bonds. Compare bands under the sample preparation conditions used. These features suggest possibilities to test but do not identify an unexpected band by themselves.
Boster reagents
COL5A3 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-Collagen alpha-3(V) chain COL5A3 Antibody
Cat # A10525
The catalog reports A10525, a human-reactive anti-COL5A3 polyclonal antibody, with a Western blot image described as analysis of various cells. The supplied information does not identify the cells or report blot conditions or independent validation.
Which to pick: A10525 is the only listed COL5A3 antibody. It reports human reactivity and includes a Western blot image; check the product details for sample and protocol fit before use.
Source: BosterBio COL5A3 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P25940.
- Human Protein Atlas. COL5A3 tissue expression.
- PMC13543591 — target-verified WB comparison
- PMC7359459 — target-verified WB comparison
- PMC11855316 — target-verified WB comparison