ID4 / DNA-binding protein inhibitor ID-4 · Western blot design guide

Design a Western Blot for ID4

Real validated ID4 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-ID4 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

Western blot protocol sheet for ID4: expected band ~16.6 kDa, hero antibody A03975-1, catalog values and labelled standard workflow; separate PMC comparisons on the guide — Printable **ID4** Western blot protocol sheet — expected band ~16.6 kDa, antibody A03975-1, controls and PMC citations. Open the full ID4 WB guide →

Navigate to: Protocols Band Controls Antibodies

ID4 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	~16.6 kDa
	Gel	15% (standard starting point)
	Negative control ⓘ	Suggested KO / knockdown lysate

Important caveats

Reasons your observed band may differ from the expected size.

	PTM	—
	Caveat	Peptide-blocking control
	Gene-set association	MSigDB C7 membership
	Isoform	1 isoform(s)

Section 1

Real Curated ID4 Western Blot Protocols

The A03975-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.

Recommended Western blot protocol parameters
Sample / lysate	various cells (catalog A03975-1)
Gel %	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	0.2 µm PVDF (standard starting point)
Blocking	5% milk or 5% BSA in TBST (standard starting point)
Primary antibody	A03975-1 · 1:500 (catalog A03975-1)
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 ID4 Western Blot Band Size?

ID4 is predicted to be 16.6 kDa; no empirical band size is supplied, and its listed features do not demonstrate altered migration.

What am I looking at on my blot?
Single band near 16.6 kDa	Consistent with the predicted ID4 mass; confirm its identity with controls.
Band near 16.6 kDa in a nuclear fraction	Consistent with ID4's nuclear location.
Faint or absent cytoplasmic band	Consistent with ID4's nuclear location.
Band far from 16.6 kDa	Its identity is uncertain from the supplied ID4 features.

💡Expected ID4 appearanceID4 has a predicted mass of 16.6 kDa, with no empirical band size supplied; confirm a band near that size using antibody specificity and fractionation controls.

How each factor affects band size
UniProt predicted molecular mass	Places ID4 near 16.6 kDa by sequence prediction.
Full-length ID4 polypeptide mass	Corresponds to 16,622 Da before any unlisted changes.
161-residue ID4 sequence	Underlies the predicted mass but does not establish empirical migration.
Sequence-based size estimate	Provides a reference near 16.6 kDa, not a validated band position.

Why is my band missing or off?
Situation	Likely cause	Next action
No band in lysate	Nuclear ID4 may be poorly recovered in the preparation.	Check nuclear extraction and loading with a nuclear marker.
Band higher than expected	The supplied features do not establish a migration shift.	Check antibody specificity against a positive control.
Band lower than expected	The supplied features do not establish a smaller ID4 form.	Check specificity and sample integrity.
Multiple bands	The supplied features do not establish multiple ID4 isoforms.	Use peptide competition or an independent antibody to identify the ID4 band.
Weak or no signal	Nuclear ID4 may be diluted in whole-cell lysate.	Try a nuclear fraction and verify extraction and loading.

Source: UniProt P47928 Human Protein Atlas product datasheet (A03975-1)

Experiment evidence

Western blot images showing ID4 band

A03975-1 · ID4

← swipe / scroll for more images →

Real WB data behind the band-size expectations in this section.

Sample controls for ID4 Western blot

🧪For positive controls for ID4 in Western blot, you can use no HPA-validated tissue or cell line 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: ID4 is nuclear, but the supplied HPA data cannot establish a positive or negative tissue control.

HPA tissue expression evidence for ID4

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 ID4 Western Blot Tips

Deeper troubleshooting and optimisation questions for ID4, answered from its protein features.

Where should the ID4 band appear relative to its predicted mass?

Band shift · ID4 has a predicted mass of 16.6 kDa. Use this as a starting point for identifying the band; no observed band position is supplied, so an exact apparent mass cannot be specified.

Could annotated isoforms explain multiple ID4 bands?

Isoforms · The supplied record lists one isoform and no alternative sequence. It therefore provides no isoform-based explanation for multiple bands. Evaluate other bands before assigning them to ID4.

Do annotated modifications predict an ID4 band shift?

PTM · No modified residues or glycosylation sites are listed. The supplied features do not support assigning a shifted band to a specific modification, and they do not establish whether a visible shift occurs.

Does this guide establish induction of ID4?

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.

What transfer method to use for ID4 Western blot?

Transfer · ID4 is predicted to be 16.6 kDa. Choose and check transfer conditions for retention of a small protein, then confirm recovery on the membrane. The supplied features do not identify a specific transfer method.

How should blocking be optimized?

Blocking · Standard workflow guidance: follow the A03975-1 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 ID4 be quantified across samples?

Quantitation · ID4 is listed as nuclear. For comparisons, use the same sample preparation and nuclear enrichment approach across samples so differences in nuclear recovery do not confound band intensity.

Could ID4 binding partners affect band interpretation?

Interpretation · ID4 forms heterodimers with other HLH proteins. Keep that interaction in mind when interpreting results from assays that preserve protein complexes; the supplied features do not establish that a complex produces a Western blot band.

Compare them with the 16.6 kDa predicted mass, but do not assign identity from size alone. The record lists one isoform and no annotated modification sites or observed band position, so it does not explain a particular extra band.

Boster reagents

ID4 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 Western Blot analysis of various cells using Id4 Polyclonal Antibody diluted at 1:500 cells nucleus extracted by Minute TM Cytoplasmic and Nuclear Fractionation kit .

Western Blot analysis of various cells using Id4 Polyclonal Antibody diluted at 1:500 cells nucleus extracted by Minute TM Cytoplasmic and Nuclear Fractionation kit .

Anti-Id4 Antibody

Cat # A03975-1

View product →

the supplier A03975-1 is an anti-ID4 antibody listed for Western blotting with reported human, mouse, and rat reactivity. Its WB image caption describes nuclear extracts from various cells tested at 1:500, without identifying the cell types or species.

Which to pick: A03975-1 is the only listed option and has a WB image. Consider it for human, mouse, or rat samples based on listed reactivity; the caption documents nuclear extracts at 1:500 but does not identify the tested species.

Source: BosterBio ID4 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.

References

UniProt Consortium. UniProt entry P47928.
Human Protein Atlas. ID4 tissue expression.
PMC3145841 — target-verified WB comparison
PMC7604853 — target-verified WB comparison
PMC4196193 — target-verified WB comparison