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- Table of Contents
ITIH4 / Inter-alpha-trypsin inhibitor heavy chain H4 · Western blot design guide
Design a Western Blot for ITIH4
Source-linked ITIH4 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-ITIH4 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
ITIH4 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 | ~103.4 kDa | |
| Gel | 8–10% (standard starting point) | |
| Negative control | Suggested KO / knockdown lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated + Cleaved | |
| Caveat | Proteolytic cleavage controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 4 isoform(s) |
Section 1
Source-Linked ITIH4 Western Blot Protocol Options
The A03593 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | HEK293T, Raw264.7, PC12 (catalog A03593) |
| 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 | A03593; 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 ITIH4 Western Blot Band Size?
ITIH4 has a predicted 103.4 kDa precursor; secretion, cleavage, glycosylation, and isoforms could affect its blot, but their migration effects are unmeasured here.
| Band near 103.4 kDa | Consistent with the predicted ITIH4 precursor mass; identity requires controls |
| Band below 103.4 kDa | May reflect signal-peptide or propeptide cleavage |
| Band above 103.4 kDa | May reflect glycosylation; its effect on migration is unmeasured here |
| Several bands at different positions | Could reflect isoforms 1, 2, 3, and 4 or processing; distinct migration is unproven |
| Little or no band in whole-cell lysate | ITIH4 is secreted and may be depleted from the cells |
💡Expected ITIH4 appearanceUniProt predicts a 103.4 kDa precursor, but no empirical band size is supplied; assess bands with an ITIH4-positive control and antibody-specificity controls because processing and glycosylation may alter migration.
| UniProt predicted precursor mass | Provides a 103.4 kDa sequence-based reference, not a measured band |
| N-linked glycosylation at Asn81, Asn207, Asn274, Asn517, and Asn577 | May increase apparent size; Asn274 is annotated as atypical |
| O-linked glycosylation at Thr719, Thr720, and Thr722 | May affect apparent size, with no measured shift supplied |
| Signal peptide at residues 1–28 | Cleavage yields a smaller mature product than the precursor |
| Propeptide at residues 662–688 | Removal may yield smaller processed products; fragment masses are unspecified |
| Splice isoforms 1, 2, 3, and 4 | May differ in size, but their masses and migration are unspecified |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Secreted ITIH4 may be scarce in whole-cell lysate | Test conditioned medium alongside an ITIH4-positive control |
| Band higher than expected | Annotated glycosylation may affect migration | Compare untreated and deglycosylated samples with an ITIH4-positive control |
| Band lower than expected | Signal-peptide or propeptide processing may reduce size | Compare with an ITIH4-positive control and verify recognition with a second antibody |
| Broad smear instead of sharp band | Heterogeneity at the annotated glycosylation sites is possible | Compare untreated and deglycosylated samples to test whether glycosylation contributes |
| Multiple bands | Alternative splicing or processing may contribute; distinct isoform bands are unverified | Check antibody specificity and compare bands across samples with different ITIH4 expression |
| Weak or no signal | Secretion may limit ITIH4 retained in cells | Probe conditioned medium and include an ITIH4-positive control |
| Fragments below expected size | Proteolytic cleavage is possible; kallikrein cleaves ITIH4 in vitro | Handle samples with protease inhibitors and confirm fragments with an independent ITIH4 antibody |
Experiment evidence
Western blot images showing ITIH4 band
A03593 · ITIH4
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for ITIH4 Western blot
🧪For positive controls for ITIH4 in Western blot, you can use an HPA-positive tissue or cell once identified; none is listed in the supplied evidence.
Positive control: No high/medium HPA tissue identified
Negative control: Suggested KO / knockdown lysate
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: ITIH4 is secreted, so whole-cell lysates may give weak signal; conditioned medium may be more suitable.
HPA tissue expression evidence for ITIH4
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 |
|---|---|---|---|---|
| No high/medium HPA tissues identified in the supplied evidence. | ||||
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| No lower-expression tissue rows available in the supplied evidence. | ||||
Section 3
Advanced ITIH4 Western Blot Tips
Deeper troubleshooting and optimisation questions for ITIH4, answered from its protein features.
How should ITIH4 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 ITIH4 isoforms account for multiple bands?
Isoforms · Four isoforms are listed. Isoforms 2–4 lack residues 621..650; isoform 4 also lacks 727..765 and 851..866. In isoform 2, Ala727 is replaced by ACPSCSRSRAPAVPA. Compare antibody epitope coverage with these regions before assigning bands to isoforms; the sequence differences do not prove that separate bands will resolve.
Which ITIH4 glycosylation sites matter when interpreting bands?
PTM · UniProt lists N-linked sites at Asn81, Asn207, atypical Asn274, Asn517, and Asn577, plus O-linked sites at Thr719, Thr720, and Thr722. These are UniProt canonical-sequence coordinates; antibody or paper numbering may differ. Site annotation alone cannot identify the cause of a particular band.
Does this guide establish induction of ITIH4?
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 ITIH4?
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 A03593 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 ITIH4 bands be quantified across samples?
Quantitation · Define which band or bands the antibody detects before comparing signal. ITIH4 is secreted and has four annotated isoforms, processing regions, and glycosylation sites; these features can complicate assigning one band to total ITIH4. Use the same band definition and sample fraction across comparisons.
Why might ITIH4 migrate differently from its predicted 103.4 kDa?
Interpretation · The 103.4 kDa value is predicted. ITIH4 has a signal peptide at residues 1..28, a propeptide at 662..688, and eight annotated glycosylation sites. Processing and glycosylation may affect apparent mass, but these features alone do not establish a visible shift or its size.
UniProt annotates a signal peptide at 1..28 and a propeptide at 662..688, using canonical-sequence numbering. Check whether the antibody recognizes a region retained in the proposed product. These annotations do not specify which processed species is present in a sample or its apparent mass.
Consider cleavage as one possibility: UniProt reports that C-terminal binding to DNAJC1 protects ITIH4 against kallikrein cleavage in vitro. Also consider annotated processing and isoform differences. A band’s size alone cannot establish its identity or show that kallikrein acted in the sample.
UniProt lists ITIH4 as secreted and annotates a signal peptide at residues 1..28. Include an appropriate extracellular sample fraction when investigating weak cellular signal. The feature record does not establish how much ITIH4 is present in any particular sample.
UniProt annotates a signal peptide at 1..28 and a propeptide at 662..688, using canonical-sequence numbering. Check whether the antibody recognizes a region retained in the proposed product. These annotations do not specify which processed species is present in a sample or its apparent mass.
Consider cleavage as one possibility: UniProt reports that C-terminal binding to DNAJC1 protects ITIH4 against kallikrein cleavage in vitro. Also consider annotated processing and isoform differences. A band’s size alone cannot establish its identity or show that kallikrein acted in the sample.
UniProt lists ITIH4 as secreted and annotates a signal peptide at residues 1..28. Include an appropriate extracellular sample fraction when investigating weak cellular signal. The feature record does not establish how much ITIH4 is present in any particular sample.
Boster reagents
ITIH4 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-ITIH4 Antibody
Cat # A03593
The catalog reports one anti-ITIH4 antibody, A03593, with reported human, mouse, and rat reactivity. Its Western blot image shows HEK293T, Raw264.7, and PC12 whole-cell lysates at 1:500 dilution. These are the documented test contexts.
Which to pick: A03593 is the only listed option. Consider it when its reported reactivity and the shown lysate conditions fit your experiment; the supplied evidence does not establish performance in other samples.
Source: BosterBio ITIH4 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q14624.
- Human Protein Atlas. ITIH4 tissue expression.
- PMC3192726 — target-verified WB comparison
- PMC3635902 — target-verified WB comparison
- PMC11417925 — target-verified WB comparison