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- Table of Contents
PHF1 / PHD finger protein 1 · Western blot design guide
Design a Western Blot for PHF1
Real validated PHF1 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-PHF1 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
PHF1 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 | ~62.1 kDa | |
| Observed band | ~62 kDa | |
| Gel | 5–20% (catalog A03758-2) | |
| Positive control | Appendix (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 | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated PHF1 Western Blot Protocols
The A03758-2 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | rat heart, mouse heart, mouse C2C12 (catalog A03758-2) |
| Gel % | 5–20% (catalog A03758-2) |
| Load | 30 ug; reducing conditions (catalog A03758-2) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A03758-2) |
| Membrane | nitrocellulose membrane (catalog A03758-2) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A03758-2) |
| Primary antibody | A03758-2 · 0.5 μg/mL (catalog A03758-2) |
| Primary incubation | overnight at 4°C (catalog A03758-2) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A03758-2) |
| Secondary incubation | 1.5 hour at RT (catalog A03758-2) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A03758-2) |
| Detection | ECL (catalog A03758-2) |
Section 2
What Is the Expected PHF1 Western Blot Band Size?
PHF1 is predicted at 62.1 kDa and observed at ~62 kDa; no cause for the small difference is established.
| Band at ~62 kDa | Matches the empirical PHF1 band and its 62.1 kDa predicted mass |
| Additional band at an unspecified size | Could reflect isoform 1 or 2; band identity requires confirmation |
| Stronger ~62 kDa band in a nuclear fraction | Consistent with PHF1 nuclear localization |
| No visible mobility change despite phosphorylation | Ser420 phosphorylation need not produce a resolved shift |
💡Expected PHF1 appearancePHF1 is predicted at 62.1 kDa and observed at ~62 kDa; confirm band identity with appropriate controls because isoform migration and any effect of Ser420 phosphorylation are unestablished.
| Predicted PHF1 mass | 62.1 kDa provides the sequence-based reference; the observed band is ~62 kDa |
| Isoform 1 | Its apparent size relative to isoform 2 is not supplied |
| Isoform 2 | Its apparent size relative to isoform 1 is not supplied |
| Ser420 phosphorylation | A mobility shift is possible but not established by the supplied evidence |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | PHF1 may be difficult to detect in the sampled lysate | Check sample loading and examine a nuclear fraction |
| Band higher than expected | Its identity or a modification-dependent shift is unconfirmed | Compare with a PHF1 knockdown or knockout control |
| Band lower than expected | An alternative isoform or degradation is possible | Check sample integrity and confirm the band with PHF1 depletion |
| Multiple bands | Isoforms 1 and 2 are annotated, but their migration is unknown | Use PHF1 depletion to identify specific bands |
| Weak or no signal | PHF1 abundance or recovery may be low in the sample | Check loading and test nuclear enrichment |
Experiment evidence
Western blot images showing PHF1 band
A03758-2 · PHF1
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for PHF1 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for PHF1 in Western blot, you can use appendix tissue, which shows high HPA expression.
Positive control: Appendix (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: Adipose tissue is a plausible negative control because HPA reports PHF1 as not detected there.
HPA tissue expression evidence for PHF1
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 |
|---|---|---|---|---|
| Appendix | glandular cells | High | Protein (IHC) | HPA → |
| Breast | glandular cells | High | Protein (IHC) | HPA → |
| Cerebellum | Purkinje cells | High | Protein (IHC) | HPA → |
| Cervix | squamous epithelial cells | High | Protein (IHC) | HPA → |
| Colon | endothelial cells | High | 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 → |
| Adrenal gland | glandular cells | Medium | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Medium | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | Medium | Protein (IHC) | HPA → |
| Caudate | glial cells | Medium | Protein (IHC) | HPA → |
Section 3
Advanced PHF1 Western Blot Tips
Deeper troubleshooting and optimisation questions for PHF1, answered from its protein features.
How should PHF1 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 PHF1 isoforms produce different bands?
Isoforms · Yes. Isoform 1 replaces canonical residues 350–457 and lacks canonical residues 458–567. Compare the expected sizes and antibody recognition regions for isoforms 1 and 2 before assigning additional bands.
How should phosphorylation at Ser420 be interpreted?
PTM · UniProt lists phosphoserine at canonical position 420. That position lies within the segment replaced in isoform 1, so do not assume the same site exists there. This modification alone does not establish a detectable band shift.
Does this guide establish induction of PHF1?
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 PHF1?
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 A03758-2 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 PHF1 bands be quantified across samples?
Quantitation · Measure the same assigned band and cellular fraction across samples. PHF1 is listed in the nucleus and at the centrosome, so differences between fractions may reflect localization. Account for the two isoforms if the antibody detects both.
Should PHF1 migrate above its predicted mass?
Interpretation · The supplied apparent band is approximately 62 kDa, close to the predicted 62.1 kDa. The listed features do not establish a visible mobility shift; compare samples against a molecular weight marker.
PHF1 localizes to double-strand break sites following DNA damage. If comparing damaged and untreated samples, keep the sampled cellular fraction consistent; a localization change could alter the signal in a fraction without establishing a change in total PHF1.
Check whether a band could represent isoform 1, whose C-terminal sequence differs from the 567-residue canonical sequence. The listed Ser420 phosphorylation does not by itself explain a visible shift. The supplied features provide no basis to assign an unexpected band to glycosylation.
PHF1 localizes to double-strand break sites following DNA damage. If comparing damaged and untreated samples, keep the sampled cellular fraction consistent; a localization change could alter the signal in a fraction without establishing a change in total PHF1.
Check whether a band could represent isoform 1, whose C-terminal sequence differs from the 567-residue canonical sequence. The listed Ser420 phosphorylation does not by itself explain a visible shift. The supplied features provide no basis to assign an unexpected band to glycosylation.
Boster reagents
PHF1 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-PHF1 Antibody Picoband®
Cat # A03758-2
The catalog reports one anti-PHF1 antibody, A03758-2, for Western blotting. Its WB image shows a band near 62 kDa in rat heart, mouse heart, and mouse C2C12 lysates. Human reactivity is listed, but no human WB example is supplied.
Which to pick: A03758-2 is the only listed option. Its WB image provides a starting point for rat heart, mouse heart, or C2C12 samples under the reported conditions; confirm performance in your own samples, especially human samples.
Source: BosterBio PHF1 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry O43189.
- Human Protein Atlas. PHF1 tissue expression.
- PMC3734045 — target-verified WB comparison
- PMC9363432 — target-verified WB comparison