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- Table of Contents
TNFAIP3 / Tumor necrosis factor alpha-induced protein 3 · Western blot design guide
Design a Western Blot for TNFAIP3
Real validated TNFAIP3 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-TNFAIP3 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
TNFAIP3 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 | ~89.6 kDa | |
| Gel | 8–10% (standard starting point) | |
| Positive control | Lung (IHC candidate; verify WB) +4 more | |
| Negative control | Spleen (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated + Acetylated | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated TNFAIP3 Western Blot Protocols
The A00224-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | SK-N-SH cell lysate (catalog A00224-1) |
| Gel % | 8–10% (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 | A00224-1 · 1 μg/mL (catalog A00224-1) |
| 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 TNFAIP3 Western Blot Band Size?
TNFAIP3 has a predicted monomer mass of 89.6 kDa; homodimerization could affect migration, but no empirical band size or migration effect is demonstrated.
| Band near 89.6 kDa | Consistent with the predicted full-length monomer; confirm identity with antibody controls. |
| Band near 179 kDa | Could reflect a retained homodimer; its migration on this blot is unverified. |
| Bands near 89.6 and 179 kDa | Could reflect monomer and retained homodimer; confirm both bands. |
| Upper band absent under denaturing conditions | Consistent with dissociation of the documented homodimer, pending band-identity controls. |
💡Expected TNFAIP3 appearanceTNFAIP3 has a predicted monomer mass of 89.6 kDa, but no empirical band size is supplied; confirm any band near that position with ordinary identity controls.
| Predicted monomer mass | Places the full-length reference near 89.6 kDa. |
| Full-length monomer migration | May differ from the predicted mass; no empirical position is supplied. |
| Homodimer formation | Could place an intact dimer near twice the monomer mass. |
| Homodimer dissociation | Would leave a monomer-sized band if the dimer separates during sample preparation. |
| Situation | Likely cause | Next action |
|---|---|---|
| Band higher than expected | A retained homodimer is possible. | Compare denaturing conditions and confirm band identity. |
| Band lower than expected | The supplied features do not establish a smaller protein form. | Check sample integrity and confirm band identity. |
| Multiple bands | Monomer and retained homodimer are possible, but other bands remain unassigned. | Compare sample conditions and use antibody identity controls. |
| Weak or no signal | The supplied features do not establish abundance in the tested sample. | Check sample loading and antibody detection with a positive control. |
| Fragments below expected size | Sample degradation is possible; no cleavage product is documented here. | Check sample handling and confirm fragment identity. |
Experiment evidence
Western blot images showing TNFAIP3 band
A00224-1 · TNFAIP3
M00224 · TNFAIP3
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for TNFAIP3 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for TNFAIP3 in Western blot, you can use lung tissue lysate.
Positive control: Lung (IHC candidate; verify WB)
Negative control: Spleen (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside the samples.
⚠️Feasibility: Spleen is listed as not detected by HPA, but confirm its absence in the lysate used.
HPA tissue expression evidence for TNFAIP3
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 |
|---|---|---|---|---|
| Lung | macrophages | High | Protein (IHC) | HPA → |
| Ovary | ovarian stroma cells | High | Protein (IHC) | HPA → |
| Adipose tissue | adipocytes | Medium | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | Medium | Protein (IHC) | HPA → |
| Appendix | lymphoid tissue | Medium | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Spleen | cells in red pulp | Not detected | Protein (IHC) | HPA → |
| Breast | adipocytes | Low | Protein (IHC) | HPA → |
| Cervix | glandular cells | Low | Protein (IHC) | HPA → |
| Duodenum | glandular cells | Low | Protein (IHC) | HPA → |
| Esophagus | squamous epithelial cells | Low | Protein (IHC) | HPA → |
Section 3
Advanced TNFAIP3 Western Blot Tips
Deeper troubleshooting and optimisation questions for TNFAIP3, answered from its protein features.
How should TNFAIP3 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 TNFAIP3 isoforms explain multiple bands?
Isoforms · The supplied record lists one isoform and no alternative sequence, so it does not support assigning additional bands to isoforms.
Which TNFAIP3 modifications matter when interpreting a blot?
PTM · UniProt lists N-acetylalanine at position 2 and phosphoserine at positions 459, 575, and 645. These are UniProt sequence coordinates; antibody or paper numbering may differ. Their presence does not establish a detectable band shift.
How should TNF stimulation affect blot comparisons?
Induction · The supplied interaction feature reports transient binding to RNF11, ITCH, and TAX1BP1 after TNF stimulation, lost after about one hour. Record stimulation time when comparing samples. This interaction evidence does not establish a change in TNFAIP3 abundance.
What transfer method to use for TNFAIP3 Western blot?
Transfer · Select and check a transfer setup suitable for a protein near 89.6 kDa. Verify transfer before interpreting a weak or absent signal; the supplied features do not establish a specific membrane or transfer mode.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A00224-1 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 TNFAIP3 bands be quantified across samples?
Quantitation · Compare samples prepared from the same cellular fraction and normalize to an appropriate loading measure. TNFAIP3 is listed in the cytoplasm, nucleus, and lysosome, so differing fraction recovery can affect comparisons.
Should TNFAIP3 migrate at its predicted 89.6 kDa?
Interpretation · Use 89.6 kDa as a reference, not an exact migration guarantee. No observed band size is supplied, and the listed modifications alone cannot establish a visible shift or explain a mass difference.
Check them against the 89.6 kDa prediction and validate their identity. The record lists one isoform, several modifications, and homodimerization, but none alone identifies an unexpected band or demonstrates that it will appear under your blot conditions.
Check them against the 89.6 kDa prediction and validate their identity. The record lists one isoform, several modifications, and homodimerization, but none alone identifies an unexpected band or demonstrates that it will appear under your blot conditions.
Boster reagents
TNFAIP3 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-TNFAIP3 Antibody
Cat # A00224-1
Real WB data
Anti-TNFAIP3/A20 Rabbit Monoclonal Antibody
Cat # M00224
Two the supplier anti-TNFAIP3 antibodies have WB images: A00224-1 in SK-N-SH lysate and M00224 in four human cell lysates. M00224 shows an approximately 82 kDa band versus a 90 kDa expected size. These examples do not establish performance across all listed species or samples.
Which to pick: Choose A00224-1 if you need listed human, mouse, or rat reactivity; its WB image uses SK-N-SH lysate at 1 μg/mL. Choose human-reactive M00224 for the documented Caco-2, HepG2, Jurkat, and K562 WB conditions at 1:1000.
Source: BosterBio TNFAIP3 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P21580.
- Human Protein Atlas. TNFAIP3 tissue expression.
- PMC12512869 — target-verified WB comparison
- PMC3198775 — target-verified WB comparison
- PMC6066659 — target-verified WB comparison