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IL1RAP / Interleukin-1 receptor accessory protein · Western blot design guide
Design a Western Blot for IL1RAP
Source-linked IL1RAP Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-IL1RAP 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
IL1RAP 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 | ~65.4 kDa | |
| Gel | 12–15% (standard starting point) | |
| Positive control | Appendix (IHC candidate; verify WB) +4 more | |
| Negative control | Adipose tissue (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated + Phosphorylated | |
| Caveat | Glycosylation-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 4 isoform(s) |
Section 1
Source-Linked IL1RAP Western Blot Protocol Options
The A02824 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | HeLa whole cell lysate (catalog A02824) |
| Gel % | 12–15% (standard starting point) |
| Load | 20–30 µg total protein per lane; optimize for abundance (standard starting point) |
| Transfer | Standard semi-dry transfer; verify efficiency (standard starting point) |
| Membrane | 0.45 µm PVDF (standard starting point) |
| Blocking | 5% milk or 5% BSA in TBST (standard starting point) |
| Primary antibody | A02824 · 1 μg/mL (catalog A02824) |
| 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 IL1RAP Western Blot Band Size?
IL1RAP's predicted precursor mass is 65.4 kDa; cleavage, N-linked glycosylation, and isoforms could affect migration, but no empirical band size is supplied.
| Band near 65.4 kDa | Consistent with the predicted precursor mass; identity requires controls |
| Band above 65.4 kDa | N-linked glycosylation may increase apparent size |
| Band below 65.4 kDa | Signal-peptide cleavage or an isoform may contribute |
| Broad band or smear | Variation in N-linked glycosylation may contribute |
| Several discrete bands | Isoforms 1, 2, 3, and 4 could contribute if they migrate differently and are recognized by the antibody |
💡Expected IL1RAP appearanceThe IL1RAP precursor has a predicted mass of 65.4 kDa; signal-peptide cleavage and N-linked glycosylation may affect migration, but no empirical band size establishes its position.
| Predicted precursor mass | 65.4 kDa is the sequence-based reference, not a validated band position |
| N-linked glycosylation at Asn 57, 107, 111, 118, 196, 209, and 299 | May increase apparent size or broaden migration; site annotations alone do not establish a shift |
| Signal peptide at residues 1–20 | Cleavage may make the mature protein smaller than the precursor |
| Isoforms 1, 2, 3, and 4 | May differ in size, but their masses and separation on a blot are not supplied |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Membrane IL1RAP may be poorly recovered from the lysate | Check membrane protein extraction and include a positive control lysate |
| Band higher than expected | N-linked glycosylation may affect migration | Compare untreated and deglycosylated samples with a positive control |
| Band lower than expected | Signal-peptide cleavage or an isoform may contribute | Check antibody epitope coverage and compare with a positive control |
| Broad smear instead of sharp band | Variation in N-linked glycosylation may contribute | Compare untreated and deglycosylated samples |
| Multiple bands | Isoforms may contribute, but band identities are unconfirmed | Check antibody specificity with an IL1RAP knockdown or knockout control |
| Weak or no signal | Membrane protein recovery or antibody recognition may be insufficient | Check extraction, antibody conditions, and a positive control lysate |
| Fragments below expected size | Sample proteolysis may produce smaller antibody-reactive bands | Use protease inhibitors and test whether bands disappear with IL1RAP knockdown or knockout |
Experiment evidence
Western blot images showing IL1RAP band
A02824 · IL1RAP
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for IL1RAP Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for IL1RAP in Western blot, you can use appendix tissue, which has medium HPA expression.
Positive control: Appendix (IHC candidate; verify WB)
Negative control: Adipose tissue (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside the samples.
⚠️Feasibility: A clean negative tissue is available, but the membrane isoform may require a membrane-enriched lysate for a strong signal.
HPA tissue expression evidence for IL1RAP
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 → |
| Bone marrow | hematopoietic cells | Medium | Protein (IHC) | HPA → |
| Caudate | neuronal cells | Medium | Protein (IHC) | HPA → |
| Cerebral cortex | neuronal cells | Medium | Protein (IHC) | HPA → |
| Lung | macrophages | Medium | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | adipocytes | Not detected | Protein (IHC) | HPA → |
| Cerebellum | cells in granular layer | Not detected | Protein (IHC) | HPA → |
| Colon | endothelial cells | Not detected | Protein (IHC) | HPA → |
| Endometrium | cells in endometrial stroma | Not detected | Protein (IHC) | HPA → |
| Liver | cholangiocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced IL1RAP Western Blot Tips
Deeper troubleshooting and optimisation questions for IL1RAP, answered from its protein features.
How should IL1RAP 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.
How could IL1RAP isoforms affect band interpretation?
Isoforms · Four isoforms are listed. Relative to the canonical sequence, isoform 2 changes residues 351–356 and lacks 357–570; isoform 3 replaces 302–570; isoform 4 replaces 449–570. Check whether the antibody epitope is retained in the isoform of interest before assigning a band.
Decide whether the measurement should include all four isoforms or a specific one. An antibody epitope within the shared region after the signal peptide and before residue 302 could support a broader comparison; epitopes in altered regions may favor particular isoforms. Quantify only bands whose identity and detection are supported by the antibody used.
Decide whether the measurement should include all four isoforms or a specific one. An antibody epitope within the shared region after the signal peptide and before residue 302 could support a broader comparison; epitopes in altered regions may favor particular isoforms. Quantify only bands whose identity and detection are supported by the antibody used.
Which glycosylation sites matter when investigating an IL1RAP band?
PTM · The annotated N-linked asparagines are at UniProt positions 57, 107, 111, 118, 196, 209, and 299. Compare untreated and deglycosylated samples if glycosylation is a plausible cause of altered migration. The site annotations alone do not predict the size or visibility of a shift.
UniProt annotates phosphoserine at position 557 of the canonical sequence. That region is missing or replaced in isoforms 2, 3, and 4, so do not transfer the coordinate to those isoforms without mapping their sequences. The annotation alone does not establish a visible band shift.
UniProt annotates phosphoserine at position 557 of the canonical sequence. That region is missing or replaced in isoforms 2, 3, and 4, so do not transfer the coordinate to those isoforms without mapping their sequences. The annotation alone does not establish a visible band shift.
Does this guide establish induction of IL1RAP?
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 IL1RAP Western blot?
Transfer · Isoform 1 is a single-pass cell-membrane protein, and its canonical sequence has a predicted mass of 65.4 kDa. Choose transfer conditions that recover a membrane protein in this size range, then check the gel and membrane for incomplete transfer. These features do not specify a validated transfer method.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A02824 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 IL1RAP 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 IL1RAP migrate differently from its predicted mass?
Interpretation · The 65.4 kDa prediction is for the 570-residue canonical sequence. IL1RAP has a signal peptide at residues 1–20 and seven annotated N-linked glycosylation sites. These features can affect the processed protein and its apparent migration, but they do not establish a particular band position; no observed band mass was supplied.
Check the antibody epitope against the four isoform sequences first. Also consider the signal peptide at 1–20 and the seven annotated N-linked glycosylation sites when evaluating migration. Compare samples with and without deglycosylation if appropriate, but do not assign an extra band to a specific feature from migration alone.
Check the antibody epitope against the four isoform sequences first. Also consider the signal peptide at 1–20 and the seven annotated N-linked glycosylation sites when evaluating migration. Compare samples with and without deglycosylation if appropriate, but do not assign an extra band to a specific feature from migration alone.
Boster reagents
IL1RAP 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-1RAcP IL1RAP Antibody
Cat # A02824
A02824 is an anti-IL1RAP antibody listed for human reactivity. Its Western blot image shows testing on HeLa whole cell lysate at 1 μg/mL. The supplied evidence covers that sample and condition only.
Which to pick: A02824 is the only listed option and has a Western blot image from HeLa whole cell lysate. Consider it for human IL1RAP work; confirm performance in your own samples and conditions.
Source: BosterBio IL1RAP gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q9NPH3.
- Human Protein Atlas. IL1RAP tissue expression.
- PMC6349174 — target-verified WB comparison
- PMC5795386 — target-verified WB comparison
- PMC7897808 — target-verified WB comparison