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- Table of Contents
IRF6 / Interferon regulatory factor 6 · Western blot design guide
Design a Western Blot for IRF6
Source-linked IRF6 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-IRF6 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
IRF6 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 | ~53.1 kDa | |
| Gel | 12–15% (standard starting point) | |
| Positive control | Appendix (IHC candidate; verify WB) +4 more | |
| Negative control | Heart muscle (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Ubl conjugation | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 2 isoform(s) |
Section 1
Source-Linked IRF6 Western Blot Protocol Options
The M01822 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | Jurkat cell lysate (catalog M01822) |
| 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 | M01822; use the WB datasheet starting dilution (standard starting point) |
| 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 IRF6 Western Blot Band Size?
IRF6 is predicted at 53.1 kDa; two isoforms could affect migration, but no empirical band size or resolved isoform pattern is supplied.
| Single band near 53.1 kDa | consistent with the predicted IRF6 mass; identity requires confirmation |
| Several bands at different positions | could include isoforms 1 and 2; their migration is not specified |
| One band despite two annotated isoforms | the isoforms may not resolve as separate bands |
| Stronger nuclear signal after activation | consistent with IRF6 translocation to the nucleus |
💡Expected IRF6 appearanceIRF6 has a predicted mass of 53.1 kDa, but no empirical band size is supplied; confirm any candidate band with an IRF6-specific identity control.
| UniProt predicted mass | sets a 53.1 kDa reference for IRF6 |
| Isoform 1 | may differ in size from isoform 2; its mass is unspecified |
| Isoform 2 | may differ in size from isoform 1; its mass is unspecified |
| Alternative splicing of isoforms 1 and 2 | could yield different band positions, but separation is unestablished |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | nuclear IRF6 may be missed by incomplete extraction | check nuclear extraction and use an IRF6-specific positive control |
| Band higher than expected | band identity or isoform migration is uncertain | compare with an IRF6 knockdown control |
| Band lower than expected | an isoform could migrate differently; its mass is unknown | verify identity with an IRF6 knockdown control |
| Multiple bands | isoforms 1 and 2 are annotated, but their band positions are unknown | check which bands respond to IRF6 knockdown |
| Weak or no signal | IRF6 may be distributed between cytoplasm and nucleus | check both fractions and confirm extraction with fraction markers |
Experiment evidence
Western blot images showing IRF6 band
M01822 · IRF6
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for IRF6 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for IRF6 in Western blot, you can use appendix tissue lysate.
Positive control: Appendix (IHC candidate; verify WB)
Negative control: Heart muscle (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside.
⚠️Feasibility: IRF6 can shift between cytoplasm and nucleus with activation, so signal in either fraction may vary.
HPA tissue expression evidence for IRF6
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 | glandular cells | Medium | Protein (IHC) | HPA → |
| Breast | glandular cells | Medium | Protein (IHC) | HPA → |
| Caudate | glial cells | Medium | Protein (IHC) | HPA → |
| Cerebellum | cells in molecular layer | Medium | Protein (IHC) | HPA → |
| Cerebral cortex | endothelial cells | Medium | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Heart muscle | cardiomyocytes | Not detected | Protein (IHC) | HPA → |
| Skeletal muscle | myocytes | Not detected | Protein (IHC) | HPA → |
| Smooth muscle | smooth muscle cells | Not detected | Protein (IHC) | HPA → |
| Soft tissue | fibroblasts | Not detected | Protein (IHC) | HPA → |
| Spleen | cells in red pulp | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced IRF6 Western Blot Tips
Deeper troubleshooting and optimisation questions for IRF6, answered from its protein features.
How should IRF6 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 IRF6 isoforms produce bands of different sizes?
Isoforms · Yes. Isoform 2 lacks residues 1–95 of the canonical sequence, so it has a shorter sequence. The supplied features do not establish its apparent Western-blot position or whether both isoforms are expressed in your sample.
An antibody recognizing canonical residues 1–95 would miss isoform 2, which lacks that region. For detecting both isoforms, choose an epitope retained after residue 95 and verify the antibody’s stated specificity.
An antibody recognizing canonical residues 1–95 would miss isoform 2, which lacks that region. For detecting both isoforms, choose an epitope retained after residue 95 and verify the antibody’s stated specificity.
Do annotated modifications prove a band shift?
PTM · The linked UniProt record describes protein features. A modification annotation alone does not demonstrate a visible shift; retain any condition or experimental qualifier attached to it.
Does this guide establish induction of IRF6?
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 IRF6?
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 M01822 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 IRF6 be quantified across cell fractions?
Quantitation · Quantify the same fraction across matched samples and account for fraction yield when comparing nuclear and cytoplasmic signal. Nuclear enrichment after activation may reflect redistribution; it does not by itself show an increase in total IRF6.
Should IRF6 run exactly at its predicted 53.1 kDa?
Interpretation · 53.1 kDa is the predicted mass of canonical IRF6. No observed band position is supplied, so use it as a reference rather than an exact migration target.
Ubl conjugation is listed as a keyword, but no modified residue or conjugation condition is specified. That feature alone cannot establish a visible shift or explain a higher band.
IRF6 is listed in the cytoplasm and nucleus, with a note that it translocates to the nucleus in response to an activating signal. If comparing activation states, collect samples at consistent times and consider separate nuclear and cytoplasmic fractions. The supplied features do not identify a specific activating treatment.
Isoform 2 is one possibility because it lacks canonical residues 1–95. Check whether the antibody epitope remains in isoform 2 and compare the band across samples. The supplied features alone cannot identify an observed band.
Ubl conjugation is listed as a keyword, but no modified residue or conjugation condition is specified. That feature alone cannot establish a visible shift or explain a higher band.
IRF6 is listed in the cytoplasm and nucleus, with a note that it translocates to the nucleus in response to an activating signal. If comparing activation states, collect samples at consistent times and consider separate nuclear and cytoplasmic fractions. The supplied features do not identify a specific activating treatment.
Isoform 2 is one possibility because it lacks canonical residues 1–95. Check whether the antibody epitope remains in isoform 2 and compare the band across samples. The supplied features alone cannot identify an observed band.
Boster reagents
IRF6 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-IRF6 Rabbit Monoclonal Antibody
Cat # M01822
The listed anti-IRF6 rabbit monoclonal antibody, M01822, reports Human, Mouse, and Rat reactivity. Its Western blot image shows IRF6 expression in Jurkat cell lysate; the supplied evidence does not document Western blot performance in other samples.
Which to pick: M01822 is the only listed option. It has a Western blot image using Jurkat cell lysate; check whether its reported reactivity and that tested sample fit your experiment.
Source: BosterBio IRF6 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry O14896.
- Human Protein Atlas. IRF6 tissue expression.
- PMC8091794 — target-verified WB comparison
- PMC6124776 — target-verified WB comparison
- PMC12891285 — target-verified WB comparison