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- Table of Contents
CXCL5 / C-X-C motif chemokine 5 · Western blot design guide
Design a Western Blot for CXCL5
Real validated CXCL5 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-CXCL5 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
CXCL5 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 | ~12 kDa | |
| Gel | 15% (standard starting point) | |
| Positive control | Caudate (IHC candidate; verify WB) +2 more | |
| Negative control | Adrenal gland (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Cleaved | |
| Caveat | TNF/TPA-treated lysate | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated CXCL5 Western Blot Protocols
The M00946 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | A549 cell lysate treated with TNF alpha and TPA (catalog M00946) |
| 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 | M00946; 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 CXCL5 Western Blot Band Size?
CXCL5 has a predicted 12 kDa precursor; signal-peptide cleavage and homodimer formation could affect migration, but no empirical band size is supplied.
| Band near 12 kDa | Consistent with the predicted full-length CXCL5 precursor; identity requires confirmation |
| Band below 12 kDa | May reflect removal of the 1–36 signal peptide |
| Band near twice the monomer size | May reflect a CXCL5 homodimer if it survives electrophoresis |
| Little or no band in whole-cell lysate | Consistent with secretion of CXCL5 |
💡Expected CXCL5 appearanceUniProt predicts a 12 kDa full-length precursor; signal-peptide cleavage may yield a smaller mature protein, but no empirical band size is supplied, so confirm band identity with appropriate controls.
| Predicted full-length mass | Places the 114-residue precursor near 12 kDa |
| Signal peptide at residues 1–36 | Its removal yields a smaller mature protein without an established apparent mass |
| Monomeric CXCL5 | Migrates below a retained homodimer |
| CXCL5 homodimer | Could appear near twice the monomer size if it survives electrophoresis |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | CXCL5 is secreted | Check conditioned medium alongside lysate |
| Band higher than expected | A CXCL5 homodimer may survive electrophoresis | Compare denaturing conditions and confirm identity with a CXCL5 control |
| Band lower than expected | The 1–36 signal peptide may have been removed | Compare with a validated CXCL5 control and check the antibody's recognized region |
| Multiple bands | Precursor, mature protein, or retained homodimer may contribute | Compare lysate and medium under denaturing conditions and verify bands with a CXCL5 control |
| Weak or no signal | Secreted CXCL5 may be scarce in the sampled lysate | Test conditioned medium and include a positive CXCL5 control |
Experiment evidence
Western blot images showing CXCL5 band
M00946 · CXCL5
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for CXCL5 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for CXCL5 in Western blot, you can use caudate tissue.
Positive control: Caudate (IHC candidate; verify WB)
Negative control: Adrenal gland (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: CXCL5 is secreted, so conditioned medium may give a clearer signal than whole-cell lysate.
HPA tissue expression evidence for CXCL5
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 |
|---|---|---|---|---|
| Caudate | neuronal cells | Medium | Protein (IHC) | HPA → |
| Cerebral cortex | glial cells | Medium | Protein (IHC) | HPA → |
| Pancreas | pancreatic endocrine cells | Medium | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adrenal gland | glandular cells | Not detected | Protein (IHC) | HPA → |
| Appendix | glandular cells | 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 → |
Section 3
Advanced CXCL5 Western Blot Tips
Deeper troubleshooting and optimisation questions for CXCL5, answered from its protein features.
How should CXCL5 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.
Are multiple CXCL5 isoforms expected?
Isoforms · The supplied UniProt features list one isoform and no alternative sequence. Do not assign multiple bands to annotated isoforms on this evidence; evaluate the signal peptide and oligomer annotations when interpreting them.
Do annotated modifications prove a band shift?
PTM · The linked UniProt record describes protein features. A modification annotation alone does not demonstrate a visible shift; retain any condition or experimental qualifier attached to it.
Does this guide establish induction of CXCL5?
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 CXCL5?
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 M00946 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 CXCL5 be quantified across samples?
Quantitation · Because CXCL5 is secreted, choose the same sample fraction for each comparison and report whether the measurement comes from conditioned medium or cell lysate. Quantifying only lysate may miss changes in extracellular CXCL5.
Why might a CXCL5 band differ from its predicted 12 kDa mass?
Interpretation · The 12 kDa prediction is for the 114-residue sequence, which includes a signal peptide at UniProt residues 1–36. Secreted CXCL5 may lack that segment. No observed band is supplied, so the size difference cannot be confirmed from these features alone.
CXCL5 is annotated as secreted. Consider examining both conditioned medium and cell lysate; a signal in medium could reflect secreted protein rather than a change in cellular abundance.
CXCL5 has two annotated disulfide bonds. Compare reducing and nonreducing samples if mobility differs between preparations. The feature annotation alone does not predict a specific band shift.
CXCL5 is annotated as both a monomer and a homodimer. A higher band could prompt a comparison under reducing and nonreducing conditions, but these annotations do not establish that a dimer survives Western blot preparation.
CXCL5 is annotated as secreted. Consider examining both conditioned medium and cell lysate; a signal in medium could reflect secreted protein rather than a change in cellular abundance.
CXCL5 has two annotated disulfide bonds. Compare reducing and nonreducing samples if mobility differs between preparations. The feature annotation alone does not predict a specific band shift.
CXCL5 is annotated as both a monomer and a homodimer. A higher band could prompt a comparison under reducing and nonreducing conditions, but these annotations do not establish that a dimer survives Western blot preparation.
Boster reagents
CXCL5 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-CXCL5 Rabbit Monoclonal Antibody
Cat # M00946
The catalog reports one anti-CXCL5 Western blot antibody, M00946, a rabbit monoclonal with reported human reactivity. Its WB image shows CXCL5 expression in A549 cell lysate treated with TNF alpha and TPA; the supplied evidence covers this tested context only.
Which to pick: M00946 is the only listed option. Choose it for a human CXCL5 WB when its reported A549 treatment context is relevant; the supplied list provides no comparison antibody or evidence for other sample conditions.
Source: BosterBio CXCL5 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P42830.
- Human Protein Atlas. CXCL5 tissue expression.
- PMC10347419 — target-verified WB comparison
- PMC11359438 — target-verified WB comparison
- PMC11673138 — target-verified WB comparison