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AIRE · Western blot design guide
Design a Western Blot for AIRE
Real validated AIRE Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-AIRE WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Last reviewed: May 2026 · Scientific review: Boster Bio technical team
AIRE 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 | ~57.7 kDa | |
| Gel | 10–12% | |
| Negative control | siRNA / KO lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated | |
| Caveat | Alternative splicing | |
| Regulation | M2 macrophage marker | |
| Isoform | 4 isoform(s) |
Section 1
Real Curated AIRE Western Blot Protocols
Literature-validated Western blot parameters for AIRE — gel percentage, transfer, blocking, antibody incubation and detection, extracted from published methods.
| Sample / lysate | human U937 , Lane 2: human RT4 , Lane 3: human Jurkat , Lane 4: human K562 , Lane 5: rat spleen , Lane 6: rat thymus , Lane 7: mouse spleen , Lane 8: mouse thymus . 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-AIRE antigen affinity purified polyclonal antibody (Catalog # PB9980) 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 AIRE at approximately 58-68 kDa. The expected band size for AIRE is at 58 kDa |
| Gel % | 10–12% |
| 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 | 58–68 kDa |
Section 2
What Is the Expected AIRE Western Blot Band Size?
AIRE runs as a single ~58 kDa monomer band by predicted mass, with occasional higher-MW homodimer or homotetramer bands if oligomers are not fully denatured.
| single band near 58 kDa in nuclear or whole-cell lysate | matches the predicted mass of full-length, unmodified AIRE monomer, which has no signal peptide, propeptide, or glycosylation |
| faint or absent band in cytoplasm-only fraction | AIRE is predominantly nuclear, concentrated in nuclear body-like dots, with only a partial cytoplasmic pool, so non-nuclear fractions give weaker signal |
| additional bands at slightly different apparent sizes | four annotated splice isoforms of AIRE can each contribute a band of distinct length relative to the canonical sequence |
| higher-molecular-weight band near 2x or 4x the monomer size | AIRE self-associates into homodimers and homotetramers; this oligomer can persist as a higher-MW band if samples are not fully denatured, even though no inter-chain disulfide is annotated |
| single sharp band with no smearing | no annotated glycosylation sites, so the protein backbone runs at a constant, unsmeared mass |
💡Expected AIRE appearanceExpect a single band near 57-58 kDa for full-length AIRE monomer under standard reducing SDS-PAGE, since it lacks a signal peptide, propeptide, and glycosylation sites; incompletely denatured samples may also show higher-order homodimer or homotetramer bands.
| Predicted mass (UniProt) | sets the baseline ~57.7 kDa monomer band expected on a reducing SDS-PAGE gel |
| Absence of glycosylation sites | keeps the band sharp and close to predicted mass rather than smeared or shifted upward |
| No signal peptide or propeptide | means there is no cleaved mature form, so the full-length sequence is what runs on the gel |
| Homodimer/homotetramer self-association (non-disulfide) | can produce higher-MW bands at roughly 2x or 4x the monomer size if lysis or reducing conditions do not fully dissociate the oligomer |
| Four annotated splice isoforms | different isoforms may run at distinct apparent sizes, producing extra bands relative to the canonical sequence |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | AIRE forms nuclear body-like punctate and filamentous structures and is chromatin/CREBBP-associated, so standard soluble lysis buffers can leave it in the insoluble or nuclear pellet fraction | use a nuclear extraction or lysis protocol with sonication and include the pellet/nuclear fraction rather than only the soluble supernatant |
| Band higher than expected | AIRE self-associates into homodimers and homotetramers through non-disulfide interactions, so incomplete denaturation or reduction can leave residual oligomer intact | boil samples fully in fresh reducing sample buffer and confirm the gel is run under denaturing, not native, conditions |
| Multiple bands | four annotated splice isoforms of AIRE can each contribute a band of slightly different apparent size | compare against isoform-specific positive controls or use an antibody targeting a region common to all isoforms to confirm identity |
| Weak or no signal | AIRE expression is largely restricted to thymic medullary epithelial cells and a few other tissues, so bulk lysates from non-expressing lines show little protein | use thymus tissue or an AIRE-expressing mTEC line as positive control and increase total protein loaded |
| Broad smear instead of sharp band | over-loading or incomplete lysis of chromatin-associated nuclear material can trail into a smear even though AIRE itself has no glycosylation to cause heterogeneity | reduce loading amount, ensure complete sample denaturation, and add a nuclease digestion step to clear DNA-associated smearing |
Experiment evidence
Western blot images showing AIRE band
PB9980 · AIRE
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for AIRE Western blot
🧪For positive controls for AIRE in Western blot, you can use HEK293 cells transiently transfected with AIRE (recombinant overexpression lysate), since no Human Protein Atlas expression data is available to identify a confirmed endogenous positive tissue or cell line.
Positive control: AIRE-transfected HEK293 lysate
Negative control: ubiquitously expressed; use siRNA knockdown or KO line
Loading controls: Run GAPDH, β-actin, and a total-protein stain (e.g., stain-free gel, Ponceau S, or REVERT) alongside as loading controls.
⚠️Feasibility: With no HPA tissue/cell data and a nuclear/cytoplasmic localization, endogenous AIRE signal in whole-cell lysate may be low or inconsistent, so an overexpression lysate plus siRNA/KO validation is the more feasible way to establish specific controls.
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 AIRE Western Blot Tips
Deeper troubleshooting and optimisation questions for AIRE, answered from its protein features.
Why might AIRE run above its 57.7 kDa predicted mass?
AIRE is a phosphoprotein; phosphorylation adds mass and can slow SDS-PAGE migration relative to the 57.7 kDa prediction. It also forms homodimers and homotetramers, so incomplete denaturation can leave oligomeric species migrating as higher-molecular-weight bands roughly two- or four-fold above the monomer.
How many AIRE isoforms could appear on the blot?
UniProt lists four AIRE isoforms from alternative splicing. Isoforms may differ by a few kDa from the canonical 57.7 kDa monomer, so expect closely spaced bands rather than a single sharp band, depending on which isoform the antibody epitope and the tissue or cell line express.
What blocking approach suits AIRE's zinc-finger domains?
AIRE contains zinc-finger and metal-binding regions; avoid EDTA in blocking or wash buffers if probing for conformation-dependent epitopes, since it can chelate the bound zinc. Standard 5% BSA or non-fat milk blocking works for linear-epitope antibodies; no glycosylation or disulfide bonds are annotated, so harsh reducing conditions are not a concern.
What transfer method to use for AIRE Western blot?
Given AIRE's predicted 57.7 kDa mass and its homodimer/homotetramer assembly, standard wet or semi-dry transfer at 100V for 60-90 minutes suffices for the monomer; use lower voltage over a longer duration to fully transfer any higher-molecular-weight oligomeric species retained near the well.
How should AIRE band intensity be quantitated reliably?
Because AIRE is phosphorylated and forms oligomers, quantify only the band matching the expected ~57.7 kDa monomer rather than total lane intensity, and normalize to a loading control run on the same membrane to correct for transfer-efficiency differences between monomeric and oligomeric species.
What explains extra bands above the AIRE monomer?
Higher-molecular-weight bands likely reflect AIRE's native homodimer or homotetramer assembly incompletely dissociated by SDS-PAGE, or co-migration with interacting partners such as CREBBP or histone H3. Bands slightly smaller than the monomer may correspond to one of the four annotated splice isoforms rather than degradation.
Should AIRE blots use nuclear or whole-cell lysate?
AIRE localizes predominantly to the nucleus but is also detected in the cytoplasm, forming nuclear body-like dots. Whole-cell lysate captures both pools; if comparing subcellular distribution, prepare separate nuclear and cytoplasmic fractions, since a purely cytoplasmic prep could underrepresent the dominant nuclear signal.
Boster reagents
Best AIRE Western Blot Antibodies
BosterBio's AIRE 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-AIRE Antibody Picoband®
Cat # PB9980
For AIRE Western blotting, we recommend antibodies selected for top performance, extensive literature citation, and rigorous validation, including orthogonal cross-checks against negative-control tissues and complementary detection methods, ensuring confident, reproducible band identification.
Which to pick: Only one Boster AIRE antibody is catalogued, PB9980, which includes an authentic western blot validation image demonstrating specific reactivity, making it the clear choice for AIRE WB experiments.
Source: BosterBio AIRE gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry O43918.
- Human Protein Atlas. AIRE tissue expression.