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ACKR3 / Atypical chemokine receptor 3 · Western blot design guide
Design a Western Blot for ACKR3
Source-linked ACKR3 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-ACKR3 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
ACKR3 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 | ~41.5 kDa | |
| Observed band | Approximately 38 kDa | |
| Gel | 10% (catalog M02656) | |
| Positive control | Appendix (IHC candidate; verify WB) +4 more | |
| Negative control | Adrenal gland (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated + Phosphorylated | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 1 isoform(s) |
Section 1
Source-Linked ACKR3 Western Blot Protocol Options
The M02656 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human RT4, human Hela, human A431, human MCF-7, rat C6, rat PC-12, mouse NIH/3T3, mouse Neuro-2a (catalog M02656) |
| Gel % | 10% (catalog M02656) |
| Load | 30 ug; reducing conditions (catalog M02656) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog M02656) |
| Membrane | nitrocellulose membrane (catalog M02656) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog M02656) |
| Primary antibody | M02656 · 1: 500 (catalog M02656) |
| Primary incubation | overnight at 4°C (catalog M02656) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog M02656) |
| Secondary incubation | 1.5 hour at RT (catalog M02656) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog M02656) |
| Detection | ECL (catalog M02656) |
Section 2
What Is the Expected ACKR3 Western Blot Band Size?
ACKR3 is predicted at 41.5 kDa, while antibody QC observes approximately 38 kDa; the cause of the difference is not established.
| Band near 38 kDa | Empirical ACKR3 band in reducing whole-cell lysates; identity still requires controls |
| Band near 41.5 kDa | Near the predicted mass of ACKR3 |
| Band near 83 kDa | Could reflect an ACKR3 homodimer if it survives sample preparation |
| Higher or diffuse bands | Could reflect variable occupancy of the three N-linked glycosylation sites; a visible shift is unproven |
💡Expected ACKR3 appearanceACKR3 has a predicted mass of 41.5 kDa, while antibody QC reports a band near 38 kDa in reducing whole-cell lysates; confirm band identity with appropriate controls.
| UniProt predicted mass | Places the unmodified sequence near 41.5 kDa; migration can differ |
| N-linked glycosylation at Asn13 | Could increase apparent size if occupied; its contribution is unmeasured |
| N-linked glycosylation at Asn22 | Could increase apparent size if occupied; its contribution is unmeasured |
| N-linked glycosylation at Asn39 | Could increase apparent size if occupied; its contribution is unmeasured |
| ACKR3 homodimer | Could produce a band near twice the monomer mass if the dimer survives sample preparation |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Membrane and endosomal localization may limit recovery in the sampled lysate | Check membrane or endosomal fractions and an ACKR3-positive lysate |
| Band higher than expected | ACKR3 can homodimerize; N-linked glycosylation may also affect migration | Compare reducing and non-reducing samples and verify identity with an ACKR3 control |
| Band lower than expected | The reported 38 kDa band runs below the 41.5 kDa prediction; the cause is unestablished | Compare with the reported 38 kDa positive control and test band specificity |
| Broad smear instead of sharp band | Variable N-linked glycosylation is possible but has not been shown to cause a smear | Compare treated and untreated samples and confirm ACKR3 specificity |
| Multiple bands | A surviving homodimer or differently glycosylated ACKR3 could contribute | Compare reducing conditions and confirm each band with an ACKR3-specific control |
| Weak or no signal | ACKR3 resides at the membrane and predominantly in endocytic vesicles | Check sample recovery and enrich the membrane or endosomal fraction |
Experiment evidence
Western blot images showing ACKR3 band
M02656 · ACKR3
A30784 · ACKR3
A30784 · ACKR3
A02656-2 · ACKR3
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for ACKR3 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for ACKR3 in Western blot, you can use appendix tissue, which HPA reports at medium expression.
Positive control: Appendix (IHC candidate; verify WB)
Negative control: Adrenal gland (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside the samples.
⚠️Feasibility: ACKR3 is a multi-pass membrane protein that predominantly localizes to endocytic vesicles, so membrane enrichment may improve detection.
HPA tissue expression evidence for ACKR3
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 | lymphoid tissue | Medium | Protein (IHC) | HPA → |
| Bronchus | ciliated cells (ciliary rootlets) | Medium | Protein (IHC) | HPA → |
| Fallopian tube | ciliated cells (cell body) | Medium | Protein (IHC) | HPA → |
| Kidney | cells in tubules | Medium | Protein (IHC) | HPA → |
| Pancreas | exocrine glandular 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 → |
| Bone marrow | hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Caudate | glial cells | Not detected | Protein (IHC) | HPA → |
| Cerebellum | cells in granular layer | Not detected | Protein (IHC) | HPA → |
| Cerebral cortex | endothelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced ACKR3 Western Blot Tips
Deeper troubleshooting and optimisation questions for ACKR3, answered from its protein features.
How should ACKR3 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 ACKR3 bands explained by listed isoforms?
Isoforms · Only one isoform is supplied, with no alternative sequence. Do not assign additional bands to ACKR3 isoforms on this evidence alone; check band specificity before interpreting them.
Could glycosylation affect ACKR3 band migration?
PTM · UniProt lists N-linked glycosylation at Asn13, Asn22, and Asn39. Compare untreated and deglycosylated samples if you need to assess its effect on migration. These sites alone do not establish a visible shift or explain the approximately 38 kDa band.
UniProt lists phosphoserines at positions 347, 350, and 355 in its 362-residue sequence. Compare matched samples with and without phosphatase treatment if phosphorylation is the question. These coordinates use UniProt numbering and may differ from paper or antibody numbering; the listed sites do not establish a visible shift.
UniProt lists phosphoserines at positions 347, 350, and 355 in its 362-residue sequence. Compare matched samples with and without phosphatase treatment if phosphorylation is the question. These coordinates use UniProt numbering and may differ from paper or antibody numbering; the listed sites do not establish a visible shift.
Does this guide establish induction of ACKR3?
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 ACKR3 Western blot?
Transfer · ACKR3 is a multi-pass membrane protein with an apparent band near 38 kDa. Optimize transfer for recovery of that band and check transfer with total-protein staining. The supplied features do not specify a membrane type or transfer setting.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the M02656 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 ACKR3 be quantified?
Quantitation · Standard workflow guidance: quantify only a validated target band within the linear exposure range. Use consistent sample preparation and loading, retain biological replicates, and avoid interpreting saturation or loading differences as regulation.
Why might ACKR3 appear near 38 kDa instead of 41.5 kDa?
Interpretation · 41.5 kDa is the predicted mass; approximately 38 kDa is the reported apparent band. ACKR3 is a multi-pass membrane protein, so compare the observed band with the expected migration in your own samples. The listed features do not establish the cause of this difference.
ACKR3 localizes to the cell membrane and endosomes, predominantly in endocytic vesicles; ligand stimulation is associated with internalization. Compare equivalent sample fractions and normalize to total protein in that fraction, since a membrane-only measurement may change with redistribution.
ACKR3 can form homodimers and heterodimers with CXCR4, and one disulfide bond is listed. Check band specificity and compare reducing and nonreducing conditions before proposing a complex. These features do not show that a particular higher band contains a dimer.
ACKR3 localizes to the cell membrane and endosomes, predominantly in endocytic vesicles; ligand stimulation is associated with internalization. Compare equivalent sample fractions and normalize to total protein in that fraction, since a membrane-only measurement may change with redistribution.
ACKR3 can form homodimers and heterodimers with CXCR4, and one disulfide bond is listed. Check band specificity and compare reducing and nonreducing conditions before proposing a complex. These features do not show that a particular higher band contains a dimer.
Boster reagents
ACKR3 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-CXCR7 ACKR3 Rabbit Monoclonal Antibody
Cat # M02656
Real WB data
Anti-CXCR7 ACKR3 Antibody
Cat # A30784
Real WB data
Anti-GPCR RDC1/CXCR-7/ACKR3 Antibody Picoband®
Cat # A02656-2
Three the supplier anti-ACKR3 antibodies have WB images. M02656 shows a band near 38 kDa in named human, rat, and mouse cell lysates; A02656-2 shows a 41 kDa band in human lysates. A30784’s caption gives no sample identities or band size.
Which to pick: For the documented human, rat, or mouse cell lysates, consider M02656; for human RT4 or HeLa lysates, A02656-2 also has a detailed WB example. A30784 lists monkey reactivity, but its WB caption does not identify the tested cells.
Source: BosterBio ACKR3 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P25106.
- Human Protein Atlas. ACKR3 tissue expression.
- PMC6949054 — target-verified WB comparison
- PMC13010297 — target-verified WB comparison
- PMC11760809 — target-verified WB comparison