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- Table of Contents
IL9 / Interleukin-9 · Western blot design guide
Design a Western Blot for IL9
Source-linked IL9 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-IL9 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
IL9 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 | ~15.9 kDa | |
| Gel | 15% (standard starting point) | |
| Negative control | Suggested KO / knockdown lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated + Cleaved | |
| Caveat | Glycosylation-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 1 isoform(s) |
Section 1
Source-Linked IL9 Western Blot Protocol Options
The A02925-1 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 | A02925-1 · 1 μg/mL (catalog A02925-1) |
| Primary incubation | 1h (catalog A02925-1) |
| 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 IL9 Western Blot Band Size?
IL9 has a predicted 15.9 kDa precursor; signal-peptide cleavage and N-linked glycosylation could alter migration, but no native band size is demonstrated.
| Band near 15.9 kDa | consistent with the predicted precursor mass; confirm identity with controls |
| Band below 15.9 kDa | may reflect removal of the 1–18 signal peptide |
| Band above 15.9 kDa or diffuse signal | may reflect N-linked glycosylation at Asn50, Asn63, Asn78, or Asn114 |
| Little or no band in whole-cell lysate | consistent with secretion of IL9 |
💡Expected IL9 appearanceUniProt predicts a 15.9 kDa precursor; signal-peptide removal and N-linked glycosylation may alter migration, but no native band size is established, so confirm candidate bands with appropriate identity controls.
| UniProt predicted precursor mass | provides a 15.9 kDa reference, not a validated blot position |
| Signal peptide at residues 1–18 | cleavage may yield a smaller mature protein |
| N-linked glycosylation at Asn50 | may increase apparent size if the site is occupied |
| N-linked glycosylation at Asn63 | may increase apparent size if the site is occupied |
| N-linked glycosylation at Asn78 | may increase apparent size if the site is occupied |
| N-linked glycosylation at Asn114 | may increase apparent size if the site is occupied |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | IL9 is secreted | test conditioned medium alongside lysate |
| Band higher than expected | N-linked glycosylation may alter migration | compare matched samples before and after N-glycan removal |
| Band lower than expected | the signal peptide may have been removed | compare with a characterized IL9 control and verify antibody specificity |
| Broad smear instead of sharp band | heterogeneous N-linked glycosylation is possible | compare matched samples before and after N-glycan removal |
| Multiple bands | signal-peptide processing or differing N-linked glycosylation may contribute | compare conditioned medium and lysate with a specific IL9 control |
| Weak or no signal | secreted IL9 may be scarce in the tested sample | test conditioned medium and include a positive IL9 control |
Experiment evidence
Western blot images showing IL9 band
A02925-1 · IL9
A02925-1 · IL9
A02925-1 · IL9
M02925-1 · IL9
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for IL9 Western blot
🧪For positive controls for IL9 in Western blot, you can use no HPA-supported sample because no positive candidate was supplied.
Positive control: No high/medium HPA tissue identified
Negative control: Suggested KO / knockdown lysate
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: IL9 is secreted, so conditioned medium may provide a better signal than whole-cell lysate.
HPA tissue expression evidence for IL9
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 |
|---|---|---|---|---|
| No high/medium HPA tissues identified in the supplied evidence. | ||||
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| No lower-expression tissue rows available in the supplied evidence. | ||||
Section 3
Advanced IL9 Western Blot Tips
Deeper troubleshooting and optimisation questions for IL9, answered from its protein features.
How should IL9 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.
Could annotated IL9 isoforms explain multiple bands?
Isoforms · Only one isoform is supplied, with no alternative sequence. Do not assign multiple bands to IL9 isoforms from this record. Evaluate the annotated signal peptide and glycosylation sites, then confirm band identity experimentally.
Which glycosylation sites matter when interpreting IL9 bands?
PTM · UniProt annotates N-linked glycosylation at Asn50, Asn63, Asn78, and Asn114. Consider these sites when evaluating a broad band or multiple bands, but their presence alone does not establish site occupancy or explain a particular band position.
UniProt lists pyrrolidone carboxylic acid at residue 19, immediately after the annotated signal peptide at residues 1–18. These are UniProt sequence coordinates; antibody or paper numbering may differ. This modification alone does not establish a visible band shift.
UniProt lists pyrrolidone carboxylic acid at residue 19, immediately after the annotated signal peptide at residues 1–18. These are UniProt sequence coordinates; antibody or paper numbering may differ. This modification alone does not establish a visible band shift.
Does this guide establish induction of IL9?
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 IL9?
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 A02925-1 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 IL9 bands be quantified?
Quantitation · Use the same sample fraction and band region across comparisons. Because IL9 is secreted and has four annotated N-linked glycosylation sites, keep extracellular and cellular measurements distinct and account consistently for any broad or multiple signals.
Why might IL9 migrate differently from its predicted mass?
Interpretation · The predicted mass is 15.9 kDa for the 144-residue sequence. IL9 has a signal peptide at residues 1–18 and four annotated N-linked glycosylation sites. These features may affect the detected form and its migration, but the record provides no observed band or shift size.
IL9 is annotated as secreted and has a signal peptide at residues 1–18. Include the extracellular fraction, such as conditioned medium, when selecting samples; a cell lysate alone may miss secreted IL9.
Check whether the bands are compatible with a secreted protein bearing a signal peptide at residues 1–18 and N-linked sites at Asn50, Asn63, Asn78, and Asn114. The supplied features cannot identify an unexpected band or prove that processing or glycosylation caused its position; confirm its identity experimentally.
IL9 is annotated as secreted and has a signal peptide at residues 1–18. Include the extracellular fraction, such as conditioned medium, when selecting samples; a cell lysate alone may miss secreted IL9.
Check whether the bands are compatible with a secreted protein bearing a signal peptide at residues 1–18 and N-linked sites at Asn50, Asn63, Asn78, and Asn114. The supplied features cannot identify an unexpected band or prove that processing or glycosylation caused its position; confirm its identity experimentally.
Boster reagents
IL9 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-IL-9 Antibody
Cat # A02925-1
Real WB data
Anti-IL9 Rabbit Monoclonal Antibody
Cat # M02925-1
Two the supplier anti-IL9 antibodies are listed for human reactivity, each with a Western blot image. A02925-1 was shown on human spleen tissue lysate; M02925-1 was shown with recombinant human IL9 protein cell lysate. No broader sample validation is supplied.
Which to pick: For human spleen tissue lysate, choose A02925-1, which has a WB image using 15 μg per lane and 1 μg/mL antibody. M02925-1 has a WB image with recombinant human IL9 protein cell lysate. Both list human reactivity.
Source: BosterBio IL9 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P15248.
- Human Protein Atlas. IL9 tissue expression.
- PMC9065342 — target-verified WB comparison