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IFIH1 / Interferon-induced helicase C domain-containing protein 1 · Western blot design guide
Design a Western Blot for IFIH1
Real validated IFIH1 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-IFIH1 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
IFIH1 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 | ~116.7 kDa | |
| Gel | 8–10% (standard starting point) | |
| Positive control | Duodenum (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 | Phosphorylated + Ubl conjugation | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated IFIH1 Western Blot Protocols
The A00263 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | Daudi cell lysate (catalog A00263) |
| Gel % | 8–10% (standard starting point) |
| Load | 20–30 µg total protein per lane; optimize for abundance (standard starting point) |
| Transfer | Wet/tank transfer; optimize duration (standard starting point) |
| Membrane | 0.45 µm PVDF (standard starting point) |
| Blocking | 5% milk or 5% BSA in TBST (standard starting point) |
| Primary antibody | A00263 · (A) 1, (B) 2 and (C) 4 μg/mL (catalog A00263) |
| 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 IFIH1 Western Blot Band Size?
IFIH1 is predicted at 116.7 kDa; isoforms and dsRNA-dependent homodimers could affect patterns, but no empirical migration is supplied.
| Band near 116.7 kDa | Consistent with the predicted IFIH1 mass; confirm identity with antibody controls. |
| Band near twice the predicted mass | Could reflect a dsRNA-dependent homodimer if it survives sample preparation. |
| Several discrete bands | Isoforms 1 and 2 are possible contributors, but their migration is unknown. |
| Reduced band in a cytosolic fraction | IFIH1 can relocate to mitochondria after viral RNA stimulation and ISGylation. |
💡Expected IFIH1 appearanceUniProt predicts 116.7 kDa for IFIH1; no empirical band size is supplied, so confirm any band near that position with antibody and sample controls.
| Predicted molecular mass | Suggests a band near 116.7 kDa. |
| Splice isoform 1 | May migrate differently from isoform 2; its size is not supplied. |
| Splice isoform 2 | May migrate differently from isoform 1; its size is not supplied. |
| dsRNA-dependent homodimerization | Could produce a higher band if the homodimer survives sample preparation. |
| Situation | Likely cause | Next action |
|---|---|---|
| Band higher than expected | A dsRNA-dependent homodimer may have survived sample preparation. | Check denaturation and compare samples with different dsRNA exposure. |
| Multiple bands | Isoforms 1 and 2 may contribute, though distinct migration is unproven. | Compare isoform expression and validate bands with an independent antibody. |
| No band in lysate | IFIH1 may be depleted from a cytosolic preparation after mitochondrial translocation. | Compare cytosolic and mitochondrial fractions with whole-cell lysate. |
| Weak or no signal | The sampled fraction may contain little IFIH1 after relocalization. | Check whole-cell lysate, loading, and a positive control. |
| Fragments below expected size | Sample degradation is possible; no specific cleavage is listed. | Prepare fresh lysate with protease inhibitors and confirm band identity. |
Experiment evidence
Western blot images showing IFIH1 band
A00263 · IFIH1
A00263-2 · IFIH1
M00263-1 · IFIH1
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for IFIH1 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for IFIH1 in Western blot, you can use duodenum lysate, which HPA scores High.
Positive control: Duodenum (IHC candidate; verify WB)
Negative control: Adipose tissue (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: IFIH1 is intracellular, and HPA identifies a not-detected tissue, making tissue lysates practical controls.
HPA tissue expression evidence for IFIH1
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 |
|---|---|---|---|---|
| Duodenum | glandular cells | High | Protein (IHC) | HPA → |
| Small intestine | glandular cells | High | Protein (IHC) | HPA → |
| Thyroid gland | glandular cells | High | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Medium | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial 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 → |
| Caudate | glial cells | Not detected | Protein (IHC) | HPA → |
| Heart muscle | cardiomyocytes | Not detected | Protein (IHC) | HPA → |
| Ovary | ovarian stroma cells | Not detected | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | Low | Protein (IHC) | HPA → |
Section 3
Advanced IFIH1 Western Blot Tips
Deeper troubleshooting and optimisation questions for IFIH1, answered from its protein features.
How should IFIH1 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 an IFIH1 isoform produce a smaller band?
Isoforms · Yes. Isoform 2 replaces canonical residues 208–221 and lacks residues 222–1025, so it is much shorter than the 1,025-residue canonical protein. Check whether the antibody epitope is retained before assigning a smaller band to isoform 2.
Which phosphorylation sites matter when interpreting IFIH1 bands?
PTM · UniProt lists phosphoserines at positions 88, 289, 291, 301, 645, and 828; position 828 is phosphorylated by RIOK3. These are UniProt coordinates, which may differ from paper or antibody numbering. Their presence does not establish a visible band shift.
How should viral RNA stimulation guide IFIH1 sample selection?
Induction · Upon viral RNA stimulation and ISGylation, IFIH1 translocates from the cytosol to mitochondria. Compare matched whole-cell samples or the same cellular fraction across conditions; a change in one fraction may reflect redistribution rather than a change in total protein.
How should transfer be checked for IFIH1?
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 A00263 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 can IFIH1 be quantified across cellular fractions?
Quantitation · Measure matched fractions consistently and identify which fraction each signal represents. IFIH1 is listed in the cytoplasm, nucleus, and mitochondrion, with mitochondrial translocation after viral RNA stimulation and ISGylation. A cytoplasmic signal alone may not represent total IFIH1.
Should IFIH1 run exactly at its predicted 116.7 kDa?
Interpretation · Use 116.7 kDa as the calculated reference for canonical IFIH1. No observed band size is supplied, and the listed features alone cannot establish a visible shift or explain a difference from calculated mass.
First check antibody recognition of isoform 2 and whether sample conditions differ. IFIH1 homodimerizes with dsRNA and can form filaments on long dsRNA, but these features do not establish that higher bands survive Western-blot preparation. The supplied features do not identify an observed band size.
First check antibody recognition of isoform 2 and whether sample conditions differ. IFIH1 homodimerizes with dsRNA and can form filaments on long dsRNA, but these features do not establish that higher bands survive Western-blot preparation. The supplied features do not identify an observed band size.
Boster reagents
IFIH1 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-MDA5 IFIH1 Antibody
Cat # A00263
Real WB data
Anti-IFIH1 Antibody Picoband®
Cat # A00263-2
Real WB data
Anti-MDA5 Monoclonal Antibody
Cat # M00263-1
Three the supplier IFIH1/MDA5 antibodies have WB images: A00263 in Daudi lysate, A00263-2 in human Jurkat and Raji lysates, and M00263-1 in THP1 lysate. These captions document specific samples, not performance across every listed species.
Which to pick: For mouse samples, A00263 and A00263-2 list mouse reactivity, but no supplied WB caption shows a mouse sample. For human work, A00263-2 provides detailed WB conditions for Jurkat and Raji; A00263 shows Daudi, and M00263-1 shows THP1.
Source: BosterBio IFIH1 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q9BYX4.
- Human Protein Atlas. IFIH1 tissue expression.
- PMC12273476 — target-verified WB comparison
- PMC11260138 — target-verified WB comparison
- PMC10680824 — target-verified WB comparison