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- Table of Contents
CCL28 / C-C motif chemokine 28 · Western blot design guide
Design a Western Blot for CCL28
Source-linked CCL28 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-CCL28 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
CCL28 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 | ~14.3 kDa | |
| Gel | 15% (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 | Glycosylated + Cleaved | |
| Caveat | Glycosylation-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 2 isoform(s) |
Section 1
Source-Linked CCL28 Western Blot Protocol Options
The A05353 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | Target-positive lysate and matched negative control (standard starting point) |
| Gel % | 15% (standard starting point) |
| Load | 20–30 µg total protein per lane; optimize for abundance (standard starting point) |
| Transfer | Short semi-dry transfer; verify retention (standard starting point) |
| Membrane | 0.2 µm PVDF (standard starting point) |
| Blocking | 5% milk or 5% BSA in TBST (standard starting point) |
| Primary antibody | A05353; 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 CCL28 Western Blot Band Size?
The predicted full-length CCL28 mass is 14.3 kDa; secretion, signal-peptide cleavage, glycosylation, and isoforms could affect detection or migration, but no empirical band is supplied.
| Band near 14.3 kDa | consistent with the predicted full-length precursor, subject to identity confirmation |
| Band below 14.3 kDa | could reflect removal of the signal peptide |
| Little or no band in whole-cell lysate | consistent with secretion of CCL28 |
| Multiple bands at different positions | could reflect isoforms 1 and 2, though distinct migration is unproven |
💡Expected CCL28 appearanceUniProt predicts 14.3 kDa for the full-length precursor; signal-peptide cleavage and N-linked glycosylation may affect migration, but no empirical band size is supplied, so confirm band identity with appropriate controls.
| Predicted full-length mass | 14.3 kDa is the precursor reference size |
| Signal peptide at residues 1–19 | cleavage yields a smaller mature protein than the precursor |
| N-linked glycosylation site at Asn78 | glycosylation may change apparent size; a visible shift is not established |
| Splice isoforms 1 and 2 | may differ in size, but their masses and migration are not supplied |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | CCL28 is secreted | check conditioned medium alongside lysate |
| Band higher than expected | glycosylation at Asn78 is possible, but its effect on migration is unproven | compare with a deglycosylated sample and confirm band identity |
| Band lower than expected | the signal peptide may have been removed | compare precursor and mature-protein preparations and confirm band identity |
| Multiple bands | isoforms 1 and 2 exist, but distinct band positions are unproven | check isoform expression and validate each band with a specificity control |
| Weak or no signal | secreted CCL28 may be scarce in the sampled lysate | test concentrated conditioned medium and a positive control |
Experiment evidence
Western blot images showing CCL28 band
A05353 · CCL28
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for CCL28 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for CCL28 in Western blot, you can use appendix tissue, which has high HPA expression.
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 alongside the samples.
⚠️Feasibility: Because CCL28 is secreted, tissue lysates may give weak signal; extracellular samples may be more suitable.
HPA tissue expression evidence for CCL28
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 | glandular cells | High | Protein (IHC) | HPA → |
| Colon | glandular cells | High | Protein (IHC) | HPA → |
| Duodenum | glandular cells | High | Protein (IHC) | HPA → |
| Rectum | glandular cells | High | Protein (IHC) | HPA → |
| Small intestine | 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 → |
| Bone marrow | hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | adipocytes | Not detected | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
| Cervix | glandular cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CCL28 Western Blot Tips
Deeper troubleshooting and optimisation questions for CCL28, answered from its protein features.
How should CCL28 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.
How could CCL28 isoforms affect band interpretation?
Isoforms · UniProt lists two isoforms. In isoform 2, residues 64–127 of the canonical sequence are replaced by a different sequence. Check whether the antibody epitope lies in this region before assigning a band to either isoform. Different sequences do not, by themselves, establish distinct visible bands.
Could glycosylation explain an additional CCL28 band?
PTM · The listed N-linked glycosylation site is Asn78, numbered on the canonical UniProt precursor sequence. It lies within the region replaced in isoform 2. Glycosylation is a possible contributor to migration differences, but the site annotation alone cannot identify an additional band or establish its apparent mass.
Does this guide establish induction of CCL28?
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 CCL28?
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 A05353 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 CCL28 bands be quantified across samples?
Quantitation · Compare signals from the same validated band and sample fraction across samples. CCL28 is secreted, and its canonical sequence has an N-linked site at Asn78, so changes in sample fraction or band selection can complicate comparisons. The supplied features do not establish which band, if any, represents a particular glycosylation state.
Why might CCL28 migrate differently from its predicted 14.3 kDa?
Interpretation · The 14.3 kDa prediction refers to the full 127-residue sequence. CCL28 has a signal peptide at residues 1–19 and an N-linked glycosylation site at Asn78. Signal peptide removal changes the protein being measured, and glycosylation may affect migration; these features alone do not predict a visible shift or establish a band’s identity.
CCL28 is annotated as secreted and has a signal peptide at residues 1–19. Include conditioned medium or another relevant extracellular sample when assessing its presence; a cell lysate alone may miss secreted protein. Interpret any lysate signal in light of signal peptide processing.
UniProt lists two disulfide bonds but supplies no bond coordinates. Record whether samples were reduced when comparing blots, since preparation conditions may affect migration or antibody recognition. The disulfide annotation alone does not predict a particular band pattern.
Check the expected signal peptide processing, the canonical Asn78 glycosylation site, the isoform 2 replacement of canonical residues 64–127, and whether the antibody recognizes both isoforms. Compare the same sample fraction and preparation conditions. No empirical apparent band is supplied, so these features cannot confirm an unexpected band’s identity.
CCL28 is annotated as secreted and has a signal peptide at residues 1–19. Include conditioned medium or another relevant extracellular sample when assessing its presence; a cell lysate alone may miss secreted protein. Interpret any lysate signal in light of signal peptide processing.
UniProt lists two disulfide bonds but supplies no bond coordinates. Record whether samples were reduced when comparing blots, since preparation conditions may affect migration or antibody recognition. The disulfide annotation alone does not predict a particular band pattern.
Check the expected signal peptide processing, the canonical Asn78 glycosylation site, the isoform 2 replacement of canonical residues 64–127, and whether the antibody recognizes both isoforms. Compare the same sample fraction and preparation conditions. No empirical apparent band is supplied, so these features cannot confirm an unexpected band’s identity.
Boster reagents
CCL28 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-C-C motif chemokine 28 CCL28 Antibody
Cat # A05353
The catalog reports A05353, an anti-CCL28 antibody with reported Human and Mouse reactivity. Its Western blot image shows K562 cell lysates using a 1:1000 primary dilution overnight at 4°C. This image documents that sample and condition, not broader validation.
Which to pick: A05353 is the only listed CCL28 antibody and has a Western blot image from K562 lysates. Check whether its reported Human and Mouse reactivity and the pictured sample fit your experiment.
Source: BosterBio CCL28 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q9NRJ3.
- Human Protein Atlas. CCL28 tissue expression.
- PMC12537860 — target-verified WB comparison
- PMC10065022 — target-verified WB comparison
- PMC6781990 — target-verified WB comparison