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- Table of Contents
IRF8 / Interferon regulatory factor 8 · Western blot design guide
Design a Western Blot for IRF8
Real validated IRF8 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-IRF8 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
IRF8 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 | ~48.4 kDa | |
| Observed band | ~48 kDa | |
| Gel | 10% (catalog PA2209) | |
| 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 | Ubl conjugation | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated IRF8 Western Blot Protocols
The PA2209 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human THP-1, mouse spleen (catalog PA2209) |
| Gel % | 10% (catalog PA2209) |
| Load | 30 ug; reducing conditions (catalog PA2209) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog PA2209) |
| Membrane | nitrocellulose membrane (catalog PA2209) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog PA2209) |
| Primary antibody | PA2209 · 0.5 μg/mL (catalog PA2209) |
| Primary incubation | overnight at 4°C (catalog PA2209) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog PA2209) |
| Secondary incubation | 1.5 hour at RT (catalog PA2209) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog PA2209) |
| Detection | ECL (catalog PA2209) |
Section 2
What Is the Expected IRF8 Western Blot Band Size?
IRF8 is predicted at 48.4 kDa and observed at approximately 48 kDa; the supplied evidence does not establish a cause for the small difference.
| Band near 48 kDa in whole-cell lysate | Consistent with IRF8; confirm identity with IRF8 depletion |
| Band near 48 kDa in resting macrophage cytoplasm | Consistent with IRF8 localization at rest |
| Band near 48 kDa in the nucleus after IFN-gamma induction | Consistent with IRF8 nuclear translocation |
| Weak nuclear band in resting macrophages | Consistent with predominantly cytoplasmic localization at rest |
💡Expected IRF8 appearanceUniProt predicts 48.4 kDa, and antibody QC reports a band at approximately 48 kDa; confirm band identity with IRF8 depletion.
| UniProt predicted mass of 48.4 kDa | Places the expected IRF8 band near 48 kDa |
| Calculated molecular weight of 48,356 Da | Expresses the same predicted mass before rounding to kDa |
| 426-residue sequence underlying the predicted mass | Supports the sequence-based 48.4 kDa estimate |
| Predicted mass compared with the observed band | The 48.4 kDa estimate closely matches the approximately 48 kDa QC band |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | IRF8 may be poorly recovered from its nuclear or cytoplasmic compartment | Check extraction and examine nuclear and cytoplasmic fractions |
| Band higher than expected | The supplied features establish no size-increasing modification | Check markers and test band identity with IRF8 depletion |
| Band lower than expected | The supplied features establish no cleaved IRF8 product | Check sample integrity and test band identity with IRF8 depletion |
| Multiple bands | Only one isoform is listed; additional bands have no established identity | Use IRF8 depletion or a second antibody to identify the IRF8 band |
| Weak or no signal | IRF8 distribution between cytoplasm and nucleus changes with IFN-gamma induction | Examine both fractions and verify extraction and loading |
Experiment evidence
Western blot images showing IRF8 band
PA2209 · IRF8
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for IRF8 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for IRF8 in Western blot, you can use appendix tissue, which HPA scores as highly expressed.
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: Tissue controls are feasible, though IFN-γ stimulation can shift IRF8 from cytoplasm to nucleus.
HPA tissue expression evidence for IRF8
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 | High | Protein (IHC) | HPA → |
| Lymph node | germinal center cells | High | Protein (IHC) | HPA → |
| Skin | lymphocytes | High | Protein (IHC) | HPA → |
| Tonsil | germinal center cells | High | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | 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 → |
| Adrenal gland | glandular cells | Not detected | Protein (IHC) | HPA → |
| Breast | adipocytes | Not detected | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
| Caudate | glial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced IRF8 Western Blot Tips
Deeper troubleshooting and optimisation questions for IRF8, answered from its protein features.
How should IRF8 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 IRF8 isoforms explain multiple bands?
Isoforms · The supplied UniProt record lists one isoform and no alternative sequence. It therefore provides no isoform-based explanation for multiple bands.
Do annotated modifications predict an IRF8 band shift?
PTM · The supplied record lists no modified residues or glycosylation sites. Its Ubl conjugation keyword does not specify a site or demonstrate a visible shift. Do not assign a shifted band to a particular modification from these features alone.
How does IFN-gamma induction affect where IRF8 is detected?
Induction · In resting macrophages, IRF8 localizes in the cytoplasm and translocates to the nucleus upon IFN-gamma induction. Compare matched cytoplasmic and nuclear fractions when testing this response.
How should transfer be checked for IRF8?
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 PA2209 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 IRF8 nuclear translocation be quantified?
Quantitation · Measure IRF8 in matched nuclear and cytoplasmic fractions from resting and IFN-gamma-induced macrophages. A change in nuclear signal can reflect redistribution, so interpret it alongside the cytoplasmic signal.
How should the observed IRF8 band compare with its predicted mass?
Interpretation · IRF8 has a predicted mass of 48.4 kDa, close to the reported apparent band at about 48 kDa. Use that region to identify the expected band; the small difference does not require a modification-based explanation.
The record supports an expected band near 48 kDa but does not identify the cause of other bands. Although IRF8 has listed interaction partners, those interactions alone do not establish that an additional band is an IRF8 complex. Check whether the band tracks with IRF8 across matched samples and fractions before assigning it.
The record supports an expected band near 48 kDa but does not identify the cause of other bands. Although IRF8 has listed interaction partners, those interactions alone do not establish that an additional band is an IRF8 complex. Check whether the band tracks with IRF8 across matched samples and fractions before assigning it.
Boster reagents
IRF8 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-Interferon regulatory factor 8 IRF8 Antibody Picoband®
Cat # PA2209
PA2209 is a rabbit polyclonal anti-IRF8 antibody listed for human and mouse. Its Western blot image shows a band near the expected 48 kDa in human THP-1 cell and mouse spleen lysates. The supplied evidence covers these samples under the reported conditions.
Which to pick: PA2209 is the only listed antibody. It has a Western blot image for human THP-1 cell and mouse spleen lysates using 0.5 μg/mL primary antibody. Consider how closely your sample and conditions match those shown.
Source: BosterBio IRF8 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q02556.
- Human Protein Atlas. IRF8 tissue expression.
- PMC7291389 — target-verified WB comparison
- PMC13021693 — target-verified WB comparison
- PMC3513378 — target-verified WB comparison