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IL6R · Western blot design guide
Design a Western Blot for IL6R
Real validated IL6R Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-IL6R WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Last reviewed: May 2026 · Scientific review: Boster Bio technical team
IL6R 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 | ~51.5 kDa | |
| Observed band | 52 kDa | |
| Gel | 10–12% | |
| Negative control | siRNA / KO lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated + Cleaved | |
| Caveat | Heavy N-glycosylation | |
| Regulation | IFN-γ-induced | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated IL6R Western Blot Protocols
Literature-validated Western blot parameters for IL6R — gel percentage, transfer, blocking, antibody incubation and detection, extracted from published methods.
| Sample / lysate | human A549 . After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA 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-IL6R antigen affinity purified polyclonal antibody (Catalog # A01425-1) 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 1:10000 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 IL6R at approximately 52KD. The expected band size for IL6R is at 52KD |
| Gel % | 10–12% |
| Load | 50ug |
| 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, 1:10000 |
| Wash | TBS-0.1% Tween, 3 × 5 min |
| Detection | ECL |
| Exposure / imaging | Tanon 5200 |
| Observed band | 52 kDa |
Section 2
What Is the Expected IL6R Western Blot Band Size?
IL6R has a 51.5 kDa predicted backbone but is observed at about 52 kDa on Western blot, a modest shift driven mainly by its N- and O-linked glycosylation.
| Band at approximately 52 kDa | matches the empirically observed native IL6R band, close to the 51.5 kDa predicted backbone |
| Band running slightly above the 51.5 kDa predicted mass, sometimes appearing diffuse | carbohydrate mass from the five N-linked and one O-linked glycosylation sites adds to the polypeptide backbone |
| Broad or fuzzy band rather than one crisp line | heterogeneous glycan occupancy across the six annotated glycosylation sites produces a mix of glycoforms |
| A second band or doublet at a different apparent size | reflects the two annotated splice isoforms of IL6R, which differ in sequence and membrane topology |
| Little or no band in whole-cell lysate despite protein being present in serum or conditioned media | the secreted/soluble form of IL6R partitions to extracellular fluid rather than the cell pellet used for lysate |
| Mature band slightly smaller than a precursor-sized species | cleavage of the 19-residue N-terminal signal peptide removes mass from the immature precursor to give the mature protein |
💡Expected IL6R appearanceIL6R typically resolves as a band around 52 kDa on reducing SDS-PAGE, close to its 51.5 kDa predicted mass but modestly broadened by glycosylation at five N-linked and one O-linked sites.
| Predicted mass (51.5 kDa, 468 aa) | sets the baseline unmodified backbone size the observed band is compared against |
| N-glycosylation at Asn55, Asn93, Asn221, Asn245, and Asn350 | adds carbohydrate mass that shifts and can broaden the band above the predicted size |
| O-glycosylation at Thr352 | contributes additional heterogeneous mass on top of the N-glycan contribution |
| Signal peptide cleavage (residues 1-19) | the mature processed protein runs slightly smaller than the uncleaved precursor |
| Isoforms 1 and 2 from alternative splicing | can produce a second band or isoform-restricted detection depending on which form a lysate expresses |
| Non-glycosylated bacterial or minimally processed recombinant standard | runs lower than native IL6R because it lacks the added glycan mass |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | the soluble/secreted form of IL6R is released into extracellular fluid rather than retained in whole-cell lysate | test serum or conditioned media alongside lysate, or enrich the membrane fraction for the cell-surface isoform |
| Band higher than expected | N- and O-linked glycosylation adds carbohydrate mass above the 51.5 kDa backbone | treat the sample with PNGase F or a glycosidase and confirm the band shifts toward the predicted mass |
| Broad smear instead of sharp band | heterogeneous occupancy across the six glycosylation sites yields a mixture of glycoforms | deglycosylate the sample or run a longer, lower-percentage gradient gel to resolve a tighter band |
| Multiple bands | co-detection of isoform 1 and isoform 2, or incomplete reduction of the intrachain disulfide bonds | ensure full reduction with fresh reducing agent and compare band pattern to isoform-specific expression controls |
| Fragments below expected size | loss of the N-terminal signal peptide during processing, or proteolytic degradation during lysis | include protease inhibitors during lysis and compare fragment size to the mature versus precursor difference |
| Weak or no signal | IL6R expression is restricted mainly to hepatocytes and immune cells, so unrelated cell types show low abundance | increase total protein loaded or select a lysate from a known IL6R-expressing cell line |
Experiment evidence
Western blot images showing IL6R band
A01425-1 · IL6R
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for IL6R Western blot
🧪For positive controls for IL6R in Western blot, you can use liver tissue lysate, since hepatocytes are a principal physiological site of IL6R expression.
Positive control: Liver tissue (hepatocytes)
Negative control: ubiquitously expressed; use siRNA knockdown or KO line
Loading controls: Alongside your target blot, probe for GAPDH and β-actin and include a total-protein stain (e.g., stain-free gel, Ponceau S, or REVERT) as loading controls.
⚠️Feasibility: No HPA expression atlas data are available for IL6R to pinpoint a clean negative tissue, and since IL6R is a single-pass membrane receptor that is also proteolytically shed into a soluble form, whole-cell lysates may underrepresent it, so pair a hepatocyte-derived positive sample with an siRNA knockdown or knockout line to confirm antibody specificity.
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 IL6R Western Blot Tips
Deeper troubleshooting and optimisation questions for IL6R, answered from its protein features.
Why does IL6R run close to its predicted mass?
IL6R's predicted mass is 51.5 kDa and it typically appears near 52 kDa. The receptor carries 6 N-glycosylation sites, but glycosylation adds only modest mass here, so the observed shift stays small rather than producing the large upward smear seen in heavily glycosylated proteins.
Which IL6R isoform shows up on a membrane blot?
IL6R has 2 isoforms. Isoform 1 is a single-pass type I membrane protein and dominates whole-cell lysate blots near 52 kDa. Given the Secreted keyword alongside Cell membrane, a shorter isoform or shed ectodomain can appear as a distinct, smaller soluble species in serum or media samples.
Does N-glycosylation affect IL6R band sharpness?
With 6 predicted glycosylation sites and no other listed modified residues, glycoform heterogeneity is the main source of migration variability. Expect a moderately diffuse rather than perfectly sharp band; PNGase F treatment before loading can collapse this into a single deglycosylated species for comparison.
Best blocking agent for this glycosylated receptor?
Because IL6R is a glycoprotein with 6 glycosylation sites, milk-based blockers can introduce lectin-like background from their own glycoproteins binding the receptor's sugar chains. A BSA-based blocking buffer avoids this interaction and gives cleaner background for detecting the 52 kDa band.
What transfer method to use for IL6R Western blot?
IL6R is a single-pass type I membrane glycoprotein at roughly 52 kDa. Wet transfer with methanol-containing tank buffer for 60-90 minutes gives more consistent transfer of this moderately sized membrane protein than semi-dry systems, which can under-transfer glycosylated membrane species.
How to ensure accurate IL6R band quantitation?
Since IL6R is a membrane protein, pair it with a membrane-associated loading control rather than a cytosolic one. Confirm linear range with a lysate dilution series, and run samples under reducing conditions so the 4 disulfide bonds don't cause partial aggregation that skews band intensity.
Why might a smaller ~50 kDa band appear?
IL6R's keywords list both Cell membrane and Secreted localization, consistent with ectodomain shedding that generates soluble IL6R. Serum or conditioned-media samples can show this slightly smaller soluble fragment separate from the full-length ~52 kDa membrane-bound form seen in cell lysates.
Boster reagents
Best IL6R Western Blot Antibodies
BosterBio's IL6R 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-IL6R Antibody Picoband®
Cat # A01425-1
Our top-performing anti-IL6R antibody is extensively cited and rigorously validated, with specificity confirmed through orthogonal approaches including negative-tissue controls and complementary methods, ensuring confident, reproducible Western blot detection of IL6R across experimental systems.
Which to pick: Only one anti-IL6R antibody is catalogued, A01425-1, backed by an actual Western blot validation image showing specific detection under standard SDS-PAGE conditions—making it the straightforward, well-supported choice for your IL6R Western blot experiments.
Source: BosterBio IL6R gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P08887.
- Human Protein Atlas. IL6R tissue expression.