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- Table of Contents
IGFBP4 / Insulin-like growth factor-binding protein 4 · Western blot design guide
Design a Western Blot for IGFBP4
Real validated IGFBP4 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-IGFBP4 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
IGFBP4 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 | ~27.9 kDa | |
| Gel | 12–15% (standard starting point) | |
| Negative control | Suggested KO / knockdown lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated + Phosphorylated | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated IGFBP4 Western Blot Protocols
The A02074 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | extracts of various cell lines, (catalog A02074) |
| Gel % | 12–15% (standard starting point) |
| Load | 20–30 µg total protein per lane; optimize for abundance (standard starting point) |
| Transfer | Short semi-dry transfer; verify retention (standard starting point) |
| Membrane | 0.45 µm PVDF (standard starting point) |
| Blocking | Blocking buffer: 3% nonfat dry milk in TBST (catalog A02074) |
| Primary antibody | A02074 · 1:1000 (catalog A02074) |
| Primary incubation | Overnight at 4 °C (standard starting point) |
| Secondary antibody | Goat Anti-Rabbit IgG, 1:10000 (catalog A02074) |
| Secondary incubation | 1 h at room temperature (standard starting point) |
| Wash | 3 × 5 min in TBST (standard starting point) |
| Detection | ECL (catalog A02074) |
Section 2
What Is the Expected IGFBP4 Western Blot Band Size?
IGFBP4 has a predicted 27.9 kDa precursor; signal-peptide cleavage and Asn125 glycosylation could affect migration, but no empirical band establishes their effects.
| Band near 27.9 kDa | compatible with the predicted precursor mass; confirm identity with controls |
| Band below 27.9 kDa | could reflect removal of the 1–21 signal peptide |
| Band above 27.9 kDa | N-linked glycosylation at Asn125 could affect migration, but a visible shift is unproven |
| Little or no band in whole-cell lysate | IGFBP4 is secreted |
| Several bands, if resolved | isoforms 1 and 2 could contribute; their migration has not been established |
💡Expected IGFBP4 appearanceThe predicted 27.9 kDa mass is for the precursor; signal-peptide cleavage and Asn125 glycosylation could alter migration, but no empirical band size is supplied, so confirm band identity with controls.
| Predicted precursor mass | 27.9 kDa provides a reference, not a validated band position |
| Signal peptide at residues 1–21 | cleavage yields a mature protein smaller than the precursor |
| N-linked glycosylation at Asn125 | could alter apparent size; the site alone does not establish a visible shift |
| Splice isoforms 1 and 2 | could differ in size, but their masses and migration are not supplied |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | IGFBP4 is secreted | check conditioned medium and a positive control |
| Band higher than expected | N-linked glycosylation at Asn125 could affect migration | compare with a deglycosylated sample and confirm band identity |
| Band lower than expected | the 1–21 signal peptide may have been removed | check antibody epitope coverage and compare precursor and mature samples |
| Multiple bands | isoforms 1 and 2 could contribute, though distinct migration is unproven | use an IGFBP4 knockdown or isoform-specific control |
| Weak or no signal | secretion may leave little IGFBP4 in the sampled material | enrich conditioned medium and include a positive control |
Experiment evidence
Western blot images showing IGFBP4 band
A02074 · IGFBP4
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for IGFBP4 Western blot
🧪For positive controls for IGFBP4 in Western blot, you can use no HPA-supported positive tissue or cell from 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: IGFBP4 is secreted, so conditioned medium may yield a stronger signal than whole-cell lysate.
HPA tissue expression evidence for IGFBP4
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 IGFBP4 Western Blot Tips
Deeper troubleshooting and optimisation questions for IGFBP4, answered from its protein features.
How should IGFBP4 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 two IGFBP4 isoforms produce different bands?
Isoforms · UniProt lists isoforms 1 and 2; isoform 2 lacks canonical residues 1..100. Consider isoform identity when interpreting bands, but the features do not establish which isoforms are present in your sample or their apparent masses.
How should glycosylation be considered when interpreting IGFBP4 bands?
PTM · The listed N-linked glycosylation site is Asn125 in canonical UniProt numbering. Comparing untreated and deglycosylated samples could test whether glycosylation contributes to a band difference; the site alone does not establish a visible shift.
UniProt lists phosphoserine at canonical residue 255, modified by FAM20C. A phosphatase comparison could test whether phosphorylation contributes to an observed difference. The feature alone does not establish a detectable mobility shift.
UniProt lists phosphoserine at canonical residue 255, modified by FAM20C. A phosphatase comparison could test whether phosphorylation contributes to an observed difference. The feature alone does not establish a detectable mobility shift.
Does this guide establish induction of IGFBP4?
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 IGFBP4?
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 A02074 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 IGFBP4 be quantified?
Quantitation · Standard workflow guidance: quantify only a validated target band within the linear exposure range. Use consistent sample preparation and loading, retain biological replicates, and avoid interpreting saturation or loading differences as regulation.
Why might IGFBP4 migrate differently from its predicted 27.9 kDa mass?
Interpretation · The 27.9 kDa prediction covers the full-length sequence, including signal peptide residues 1..21. IGFBP4 is secreted and has an N-linked glycosylation site at Asn125. These features can affect apparent mass, but the supplied evidence gives no observed band or measured shift.
IGFBP4 is annotated as secreted. Check the extracellular fraction, such as conditioned medium, when choosing a Western blot sample. Its signal peptide occupies canonical residues 1..21.
Because IGFBP4 is secreted, compare matched extracellular samples using consistent collection and loading conditions. Keep the sampled fraction consistent across conditions; a band in medium need not represent the amount remaining inside cells.
Check whether the band could reflect isoform 2, which lacks canonical residues 1..100, or processing of the 1..21 signal peptide. Asn125 glycosylation, Ser255 phosphorylation by FAM20C, and ten listed disulfide bonds are also relevant. None of these features identifies an unexpected band without further testing.
IGFBP4 is annotated as secreted. Check the extracellular fraction, such as conditioned medium, when choosing a Western blot sample. Its signal peptide occupies canonical residues 1..21.
Because IGFBP4 is secreted, compare matched extracellular samples using consistent collection and loading conditions. Keep the sampled fraction consistent across conditions; a band in medium need not represent the amount remaining inside cells.
Check whether the band could reflect isoform 2, which lacks canonical residues 1..100, or processing of the 1..21 signal peptide. Asn125 glycosylation, Ser255 phosphorylation by FAM20C, and ten listed disulfide bonds are also relevant. None of these features identifies an unexpected band without further testing.
Boster reagents
IGFBP4 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-IGFBP4 Antibody
Cat # A02074
A02074 is an anti-IGFBP4 antibody listed for Western blotting, with reported human, mouse, and rat reactivity. Its WB image caption describes cell-line extracts tested at 1:1000 dilution, but does not identify the cell lines or establish validation in every listed species.
Which to pick: A02074 is the only listed option and has a WB image. Use its reported 1:1000 antibody dilution and 25 µg protein per lane as starting conditions; verify performance in your own sample.
Source: BosterBio IGFBP4 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P22692.
- Human Protein Atlas. IGFBP4 tissue expression.
- PMC2720214 — target-verified WB comparison
- PMC13399132 — target-verified WB comparison
- PMC13100485 — target-verified WB comparison