This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
CHD4 / ATP-dependent chromatin remodeler CHD4 · Western blot design guide
Design a Western Blot for CHD4
Real validated CHD4 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-CHD4 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
CHD4 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 | ~218 kDa | |
| Observed band | ~260 kDa | |
| Gel | 5–20% (catalog A01125-3) | |
| Positive control | Adrenal gland (IHC candidate; verify WB) +4 more | |
| Negative control | Suggested KO / knockdown lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated + Acetylated | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated CHD4 Western Blot Protocols
The A01125-3 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human U20S, human HEL, human K562 (catalog A01125-3) |
| Gel % | 5–20% (catalog A01125-3) |
| Load | 30 ug; reducing conditions (catalog A01125-3) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A01125-3) |
| Membrane | nitrocellulose membrane (catalog A01125-3) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A01125-3) |
| Primary antibody | A01125-3 · 0.5 μg/mL (catalog A01125-3) |
| Primary incubation | overnight at 4°C (catalog A01125-3) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A01125-3) |
| Secondary incubation | 1.5 hour at RT (catalog A01125-3) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A01125-3) |
| Detection | ECL (catalog A01125-3) |
Section 2
What Is the Expected CHD4 Western Blot Band Size?
CHD4 is predicted at 218 kDa, while antibody QC reports approximately 260 kDa; the cause of this difference is not established.
| Band near 260 kDa | Empirical CHD4 band reported in reducing whole-cell lysates; confirm identity with appropriate controls |
| Band near 218 kDa | Near the predicted CHD4 mass, but identity requires confirmation |
| Multiple nearby bands | Could reflect CHD4 isoforms 1 and 2; distinct migration is not established |
| Weak band in a cytoplasmic fraction | CHD4 is also found in the nucleus |
💡Expected CHD4 appearanceCHD4 is predicted at 218 kDa, while antibody QC reports a band near 260 kDa in reducing whole-cell lysates; the cause of the difference is unestablished, so confirm band identity with appropriate controls.
| Predicted CHD4 mass | 218 kDa by sequence; the reported empirical band is near 260 kDa |
| Isoform 1 | Its individual mass and migration are not supplied |
| Isoform 2 | Its individual mass and migration are not supplied |
| Alternative splicing | May change apparent size, but distinct bands are not demonstrated |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Nuclear CHD4 may be underrepresented in the sample | Check nuclear recovery and include a positive whole-cell lysate |
| Band higher than expected | The reported 260 kDa band exceeds the 218 kDa sequence prediction; the cause is unestablished | Compare with the reported band and verify identity by CHD4 depletion |
| Band lower than expected | Band identity or protein integrity is uncertain | Compare a positive control and verify the band by CHD4 depletion |
| Multiple bands | Isoforms 1 and 2 are annotated, but separate migration is unproven | Use CHD4 depletion and isoform-specific evidence to assign bands |
| Weak or no signal | Low CHD4 recovery or insufficient detection | Check sample loading, nuclear recovery, and a positive control |
| Fragments below expected size | Protein degradation is possible; no cleavage product is annotated | Handle samples with protease inhibitors and verify fragments by CHD4 depletion |
Experiment evidence
Western blot images showing CHD4 band
A01125-3 · CHD4
M01125 · CHD4
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for CHD4 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for CHD4 in Western blot, you can use adrenal gland tissue, which shows high HPA expression.
Positive control: Adrenal gland (IHC candidate; verify WB)
Negative control: Suggested KO / knockdown lysate
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside CHD4.
⚠️Feasibility: No tissue is listed as not detected, so use siRNA knockdown or a KO line for a negative control.
HPA tissue expression evidence for CHD4
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 |
|---|---|---|---|---|
| Adrenal gland | glandular cells | High | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | High | Protein (IHC) | HPA → |
| Bronchus | basal cells | High | Protein (IHC) | HPA → |
| Caudate | neuronal cells | High | Protein (IHC) | HPA → |
| Cerebellum | cells in molecular layer | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Liver | hepatocytes | Low | Protein (IHC) | HPA → |
| Prostate | glandular cells | Low | Protein (IHC) | HPA → |
| Adipose tissue | adipocytes | Medium | Protein (IHC) | HPA → |
| Appendix | glandular cells | Medium | Protein (IHC) | HPA → |
| Breast | adipocytes | Medium | Protein (IHC) | HPA → |
Section 3
Advanced CHD4 Western Blot Tips
Deeper troubleshooting and optimisation questions for CHD4, answered from its protein features.
How should CHD4 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 CHD4 isoforms produce different bands?
Isoforms · UniProt lists isoforms 1 and 2. In isoform 2, the arginine at canonical UniProt position 1353 is replaced by RGVCGRPRPPPMGRSTRAVGPAHLPSLPP. This sequence change could affect migration, but the features do not establish a separate visible band. Check whether the antibody recognizes both sequences; downstream residue numbering may differ between isoforms.
Which CHD4 phosphorylation sites matter when interpreting band changes?
PTM · UniProt annotates 29 modified residues, including phosphoserines at 303, 308, 309, 310 and 319, and sites around 1531–1602. These are UniProt canonical coordinates; check numbering before comparing antibody or paper sites. A phosphorylation annotation alone does not demonstrate a visible shift. Compare matched samples before attributing a changed band to phosphorylation.
UniProt lists alternate N6-acetyllysine at canonical position 1643. Check that an antibody targeting this region can recognize the modified form if detection differs between samples. This annotation alone does not establish an apparent mass change.
UniProt lists alternate N6-acetyllysine at canonical position 1643. Check that an antibody targeting this region can recognize the modified form if detection differs between samples. This annotation alone does not establish an apparent mass change.
Does this guide establish induction of CHD4?
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 CHD4 Western blot?
Transfer · CHD4 is large: 218 kDa predicted, with a supplied observed band near 260 kDa. Select and validate transfer conditions that retain signal in that high-mass range. Use a high-mass marker and check transfer efficiency before interpreting a weak or absent CHD4 band.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A01125-3 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 CHD4 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 CHD4 run near 260 kDa instead of 218 kDa?
Interpretation · CHD4 has a predicted mass of 218 kDa, while the supplied observed band is about 260 kDa. Its annotated modifications and isoforms do not establish the cause of that difference. Compare samples with the same gel conditions and confirm the band’s identity before assigning an explanation.
CHD4 is annotated in the nucleus and at centrosomes in interphase. Compare equivalent sample fractions and loading when measuring its band; changes in fraction composition could change the signal independently of total CHD4 abundance. Quantify the same verified band across samples.
First verify band identity and sample fraction. UniProt lists two isoforms, multiple modified residues, nuclear localization and interphase centrosome association, but those features do not assign an unexpected band to a particular isoform or modification. Report its apparent mass and avoid labeling its cause without supporting evidence.
CHD4 is annotated in the nucleus and at centrosomes in interphase. Compare equivalent sample fractions and loading when measuring its band; changes in fraction composition could change the signal independently of total CHD4 abundance. Quantify the same verified band across samples.
First verify band identity and sample fraction. UniProt lists two isoforms, multiple modified residues, nuclear localization and interphase centrosome association, but those features do not assign an unexpected band to a particular isoform or modification. Report its apparent mass and avoid labeling its cause without supporting evidence.
Boster reagents
CHD4 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-CHD4 Antibody Picoband®
Cat # A01125-3
Real WB data
Anti-CHD4 Rabbit Monoclonal Antibody
Cat # M01125
Two anti-CHD4 antibodies list human reactivity and show Western blot examples. A01125-3 was tested on four human cell lysates, with a band near 260 kDa versus an expected 218 kDa. M01125 shows a 293T lysate blot; its caption gives no conditions.
Which to pick: Choose A01125-3 if its documented four-lysate blot and stated conditions match your setup. Consider M01125 for a rabbit monoclonal with a 293T lysate example, though its WB caption is brief. Both list human reactivity; neither example establishes performance in every human sample.
Source: BosterBio CHD4 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q14839.
- Human Protein Atlas. CHD4 tissue expression.
- PMC8221472 — target-verified WB comparison
- PMC6418088 — target-verified WB comparison
- PMC10342000 — target-verified WB comparison