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CCL8 · Western blot design guide
Design a Western Blot for CCL8
Real validated CCL8 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-CCL8 WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Last reviewed: May 2026 · Scientific review: Boster Bio technical team
CCL8 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 | ~11.2 kDa | |
| Observed band | ~22 kDa | |
| Gel | 12–15% | |
| Negative control | siRNA / KO lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Cleaved | |
| Caveat | Homodimer formation | |
| Regulation | IFN-γ-suppressed | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated CCL8 Western Blot Protocols
Literature-validated Western blot parameters for CCL8 — gel percentage, transfer, blocking, antibody incubation and detection, extracted from published methods.
| Sample / lysate | human Jurkat , Lane 2: human 293T , Lane 3: rat small intestine , Lane 4: rat lung , Lane 5: mouse RAW264.7 , Lane 6: mouse NIH/3T3 . After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-MCP2/CCL8 antigen affinity purified polyclonal antibody (Catalog # A03237-2) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for MCP2/CCL8 at approximately 22 kDa. The expected band size for MCP2/CCL8 is at 11 kDa |
| Gel % | 12–15% |
| Load | 30 ug |
| Transfer | nitrocellulose membrane, 150 mA, 50–90 min |
| Membrane | nitrocellulose |
| Blocking | 5% non-fat milk / TBS, 1.5 h RT |
| Primary antibody | 0.5 µg/mL |
| Primary incubation | overnight at 4 °C |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 |
| Wash | TBS-0.1% Tween, 3 × 5 min |
| Detection | ECL |
| Exposure / imaging | Tanon 5200 |
| Observed band | 22 kDa |
Section 2
What Is the Expected CCL8 Western Blot Band Size?
CCL8 has an ~11.2 kDa predicted monomer but is typically observed at ~22 kDa, consistent with its known monomer/homodimer equilibrium.
| dominant band near 22 kDa | corresponds to the CCL8 homodimer, roughly double the ~11.2 kDa monomer mass, consistent with its documented monomer/homodimer equilibrium |
| fainter band near 8-11 kDa | represents the monomeric mature CCL8 chain after signal peptide removal, running near the predicted precursor mass |
| band smaller than the 11.2 kDa full-length precursor | reflects cleavage of the 23-residue signal peptide during secretion, shortening the mature chain |
| little or no band in whole-cell lysate | CCL8 is a secreted chemokine, so it is largely exported rather than retained intracellularly |
| band position shifts slightly between reducing and non-reducing gels | the two intrachain disulfide bonds compact the folded monomer, altering migration when reduction is incomplete or absent |
💡Expected CCL8 appearanceExpect a dominant band at ~22 kDa, roughly twice the ~11.2 kDa predicted monomer mass, reflecting the CCL8 homodimer, with a fainter monomer band possible near 8-11 kDa.
| Predicted precursor mass (11.2 kDa) | sets the baseline monomer size before cleavage or oligomerization are considered |
| Signal peptide cleavage (residues 1-23) | removes the signal sequence upon secretion, so the mature monomer runs smaller than the 11.2 kDa precursor |
| Monomer/homodimer equilibrium | drives the dominant band toward roughly double the monomer mass, accounting for the observed ~22 kDa band |
| Two intrachain disulfide bonds | stabilize a compact folded structure that can migrate slightly faster than fully reduced protein when reduction is incomplete |
| N-terminal pyrrolidone carboxylic acid modification | blocks the free N-terminus but adds negligible mass, so it does not meaningfully shift apparent band size |
| Secreted localization | shifts the strongest signal toward conditioned media/supernatant fractions rather than whole-cell lysate |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | CCL8 is secreted, so little protein accumulates intracellularly in whole-cell lysate | test conditioned media or supernatant fractions in addition to, or instead of, cell lysate |
| Band higher than expected | the non-covalent homodimer resists dissociation and runs near double the monomer mass | increase reducing agent concentration and heating time before loading to favor full monomer dissociation |
| Band lower than expected | the mature secreted chain lacks the 23-residue signal peptide present in the full-length precursor | compare the band against the mature-protein mass rather than the 11.2 kDa precursor mass |
| Multiple bands | monomer and homodimer species coexist in equilibrium | run parallel reducing and non-reducing gels to distinguish which band is monomer versus dimer |
| Weak or no signal | CCL8 is a low-abundance, inducible secreted chemokine with minimal expression at baseline | stimulate cells with an inducer of CCL8 expression and concentrate conditioned media before loading |
Experiment evidence
Western blot images showing CCL8 band
A03237-2 · CCL8
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for CCL8 Western blot
🧪For positive controls for CCL8 in Western blot, you can use recombinant human CCL8 protein or conditioned medium from cytokine/LPS-stimulated monocytes and macrophages, since the Human Protein Atlas has no tissue or cell line expression data on record for this secreted chemokine.
Positive control: Stimulated monocytes/macrophages (conditioned medium)
Negative control: ubiquitously expressed; use siRNA knockdown or KO line
Loading controls: Run GAPDH and β-actin antibodies alongside a total-protein stain (stain-free gel, Ponceau S, or REVERT) as loading controls.
⚠️Feasibility: As a secreted chemokine with little to no baseline expression, CCL8 is often undetectable in unstimulated whole-cell lysate, so use concentrated conditioned medium or a stimulated (e.g., LPS-treated) sample to obtain a reliable positive signal.
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.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|
Section 3
Advanced CCL8 Western Blot Tips
Deeper troubleshooting and optimisation questions for CCL8, answered from its protein features.
Why does CCL8 run near 22 kDa instead of 11 kDa?
UniProt lists CCL8 as existing in monomer-homodimer equilibrium; the ~22 kDa apparent band corresponds almost exactly to the homodimer (2 x 11.2 kDa predicted mass), so non-reducing or non-denaturing conditions can preserve the dimer while reducing SDS-PAGE conditions should favor monomer at ~11 kDa.
Should I expect multiple CCL8 isoforms on the blot?
UniProt annotates only one CCL8 isoform, so a single band is expected; extra bands more likely reflect dimer/monomer equilibrium or nonspecific antibody binding rather than genuine isoforms.
Does the N-terminal pyroglutamate affect antibody detection?
CCL8 carries one modified residue, pyrrolidone carboxylic acid, at its mature N-terminus. This blocks Edman sequencing and can mask N-terminal epitopes, so antibodies raised against the free N-terminus may underdetect native CCL8; prefer antibodies targeting internal or C-terminal epitopes.
Is CCL8 expression induced by inflammatory stimuli?
CCL8 is annotated as a cytokine with roles in chemotaxis and the inflammatory response, consistent with induction upon inflammatory challenge such as cytokine or LPS stimulation of monocytes and fibroblasts; expect low baseline signal in unstimulated cells and increased band intensity after inflammatory stimulation.
How should I optimize blocking for CCL8 blots?
CCL8 is a heparin-binding secreted protein, so it can bind nonspecifically to membranes and to polyanionic blocking or wash components; use BSA-based blocking rather than heparin- or dextran-sulfate-containing reagents, and include thorough washes to reduce background from CCL8's basic, heparin-binding surface.
What transfer method to use for CCL8 Western blot?
CCL8 is a small (~11 kDa predicted) secreted protein stabilized by two disulfide bonds, so use semi-dry or wet transfer with 0.2 µm PVDF, shortened transfer times, and low methanol to prevent blow-through; if probing for the ~22 kDa homodimer, run non-reducing gels to preserve disulfide-linked structure before transfer.
How can I reliably quantify secreted CCL8 by blot?
As a secreted, low-abundance chemokine, CCL8 is scarce in cell lysates; concentrate conditioned media (e.g., TCA or acetone precipitation) before loading, resolve on a high-percentage or gradient gel to separate the ~11 kDa monomer from the ~22 kDa dimer, and normalize to a secreted loading control rather than a cytoplasmic housekeeping protein.
What if I see both 11 and 22 kDa bands?
Both bands fit UniProt's documented monomer/homodimer equilibrium for CCL8; the two annotated disulfide bonds can stabilize an SDS-resistant dimer near 22 kDa. Boiling samples in reducing sample buffer (with DTT or beta-mercaptoethanol) should break disulfide-linked dimers and collapse signal mainly to the ~11 kDa monomer band.
Boster reagents
Best CCL8 Western Blot Antibodies
BosterBio's CCL8 antibodies are among the best-performing WB antibodies on the market — well cited, thoroughly validated, and orthogonally cross-validated against negative tissues and complementary methods.
Real WB data
Anti-MCP2/CCL8 Antibody Picoband®
Cat # A03237-2
These Boster anti-CCL8 antibodies represent our best-performing options for this target, widely cited across published studies and rigorously validated, with specificity orthogonally confirmed against negative-tissue controls and complementary detection methods for confident, reproducible Western blot results.
Which to pick: Only one anti-CCL8 antibody is catalogued here, A03237-2, and it's the clear choice: it includes an authentic Western blot validation image showing detection of MCP2/CCL8 on SDS-PAGE, giving direct visual confirmation of expected banding before you order.
Source: BosterBio CCL8 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P80075.
- Human Protein Atlas. CCL8 tissue expression.