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- Table of Contents
PHF8 / Histone lysine demethylase PHF8 · Western blot design guide
Design a Western Blot for PHF8
Source-linked PHF8 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-PHF8 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
PHF8 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 | ~117.9 kDa | |
| Observed band | ~140 kDa | |
| Gel | 8% (catalog A03288-2) | |
| Positive control | Cerebellum (IHC candidate; verify WB) +4 more | |
| Negative control | Liver (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 | 5 isoform(s) |
Section 1
Source-Linked PHF8 Western Blot Protocol Options
The A03288-2 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human A431, human Hela, human Jurkat (catalog A03288-2) |
| Gel % | 8% (catalog A03288-2) |
| Load | 30 ug; reducing conditions (catalog A03288-2) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A03288-2) |
| Membrane | nitrocellulose membrane (catalog A03288-2) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A03288-2) |
| Primary antibody | A03288-2 · 0.5 μg/mL (catalog A03288-2) |
| Primary incubation | overnight at 4°C (catalog A03288-2) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A03288-2) |
| Secondary incubation | 1.5 hour at RT (catalog A03288-2) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A03288-2) |
| Detection | ECL (catalog A03288-2) |
Section 2
What Is the Expected PHF8 Western Blot Band Size?
PHF8 is predicted at 117.9 kDa, while antibody QC reports approximately 140 kDa; the cause of the difference is not established.
| Band near 140 kDa | Empirical PHF8 band reported in reducing whole-cell lysates |
| Band near 118 kDa | Near the predicted full-length mass; confirm its identity with antibody controls |
| Several bands at different positions | Could reflect PHF8 isoforms 1–5; their migration is not established |
| Close doublet | Could reflect PHF8 phosphorylation; a visible shift is not established |
💡Expected PHF8 appearancePHF8 has a predicted mass of 117.9 kDa, while antibody QC reports a band near 140 kDa; use ordinary band-identity controls because the cause of the difference is unestablished.
| Predicted PHF8 mass | 117.9 kDa from the supplied sequence; the reported band is near 140 kDa |
| PHF8 isoform 1 | May migrate differently from other isoforms; its individual mass is not supplied |
| PHF8 isoforms 2 and 3 | May differ in size; distinct bands are not established |
| PHF8 isoforms 4 and 5 | May differ in size; distinct bands are not established |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Nuclear PHF8 may be poorly recovered | Check nuclear extraction and a positive-control lysate |
| Band higher than expected | The reported PHF8 band is near 140 kDa, above its predicted 117.9 kDa | Compare with the QC band and verify antibody specificity |
| Band lower than expected | A PHF8 isoform is possible, but its band size is unknown | Check isoform expression and verify band identity |
| Multiple bands | PHF8 has five annotated isoforms, but their migration is unknown | Compare isoform expression and test antibody specificity |
| Weak or no signal | Nuclear PHF8 may be underrepresented in the preparation | Check nuclear recovery and include a positive-control lysate |
Experiment evidence
Western blot images showing PHF8 band
A03288-2 · PHF8
M03288 · PHF8
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for PHF8 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for PHF8 in Western blot, you can use cerebellum tissue, which HPA rates highly positive.
Positive control: Cerebellum (IHC candidate; verify WB)
Negative control: Liver (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside the samples.
⚠️Feasibility: PHF8 is nuclear and chromatin-associated, so adequate nuclear protein extraction is important.
HPA tissue expression evidence for PHF8
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 |
|---|---|---|---|---|
| Cerebellum | cells in molecular layer | High | Protein (IHC) | HPA → |
| Cerebral cortex | glial cells | High | Protein (IHC) | HPA → |
| Colon | glandular cells | High | Protein (IHC) | HPA → |
| Esophagus | squamous epithelial cells | High | Protein (IHC) | HPA → |
| Kidney | cells in tubules | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Liver | cholangiocytes | Not detected | Protein (IHC) | HPA → |
| Oral mucosa | squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Low | Protein (IHC) | HPA → |
| Lymph node | germinal center cells | Low | Protein (IHC) | HPA → |
| Ovary | follicle cells | Low | Protein (IHC) | HPA → |
Section 3
Advanced PHF8 Western Blot Tips
Deeper troubleshooting and optimisation questions for PHF8, answered from its protein features.
How should PHF8 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.
Which PHF8 isoforms could affect antibody detection?
Isoforms · Five isoforms are listed. Isoforms 2, 4 and 5 lack residues 1–36; isoforms 3 and 4 lack 478–578; isoform 5 lacks 932–1060 and has substitutions at 717–746 and 920–931. Check whether the antibody epitope is present in each isoform you intend to detect. These sequence differences do not establish where each isoform will migrate.
Which phosphorylation sites matter when interpreting PHF8 bands?
PTM · Using the supplied UniProt coordinates, CDK1 phosphorylates Ser69 and Ser120. Other listed sites are Ser651, Tyr704, Thr705, Thr706, Ser722, Ser804, Ser826, Ser834, Ser854, Ser857 and Ser880. Record the coordinate convention when comparing antibodies or papers. The site list does not show that phosphorylation produces a resolvable band shift.
Does this guide establish induction of PHF8?
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 PHF8 Western blot?
Transfer · The features do not specify a transfer method. PHF8 has a predicted mass of 117.9 kDa and an observed band near 140 kDa, so check transfer efficiency in that mass range with a molecular weight marker and a validated PHF8 control. Select transfer settings based on that result.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A03288-2 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 PHF8 be quantified across samples?
Quantitation · PHF8 is listed in the nucleus and nucleolus, with cell-cycle-dependent chromatin association. Compare like sample preparations and cell-cycle conditions, and quantify the same validated PHF8 band in each lane. Changes in a chromatin fraction need not represent changes in total PHF8 abundance.
Why might PHF8 appear near 140 kDa instead of 117.9 kDa?
Interpretation · The canonical sequence predicts 117.9 kDa, while the supplied Western blot observation is about 140 kDa. PHF8 has alternative isoforms and listed phosphorylation sites, but those features alone do not explain the apparent mass difference or establish a visible shift. Use a molecular weight marker and a validated PHF8 control to identify the band.
PHF8 is recruited to H3K4me3 sites on chromatin during interphase and dissociates from chromatin when cells enter mitosis. CDK1 phosphorylation is listed at Ser69 and Ser120. Match cell-cycle conditions when comparing samples, especially chromatin fractions, and record whether the assay measures total or fractionated PHF8.
Check antibody epitope coverage against the five isoforms and their listed sequence changes, then compare bands with a validated PHF8 control. The observed band near 140 kDa differs from the canonical predicted mass of 117.9 kDa. Isoforms and phosphorylation are possible considerations, but the supplied features alone cannot assign an unexpected band to either.
PHF8 is recruited to H3K4me3 sites on chromatin during interphase and dissociates from chromatin when cells enter mitosis. CDK1 phosphorylation is listed at Ser69 and Ser120. Match cell-cycle conditions when comparing samples, especially chromatin fractions, and record whether the assay measures total or fractionated PHF8.
Check antibody epitope coverage against the five isoforms and their listed sequence changes, then compare bands with a validated PHF8 control. The observed band near 140 kDa differs from the canonical predicted mass of 117.9 kDa. Isoforms and phosphorylation are possible considerations, but the supplied features alone cannot assign an unexpected band to either.
Boster reagents
PHF8 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-PHF8 Antibody Picoband®
Cat # A03288-2
Real WB data
Anti-PHF8 Monoclonal Antibody
Cat # M03288
The catalog reports two anti-PHF8 antibodies with Western blot images: a polyclonal tested on human cell lysates and a monoclonal tested on human, rat, and mouse samples. Reported bands were approximately 140 and 150 kDa, respectively, versus an expected 118 kDa; identity needs confirmation.
Which to pick: Choose A03288-2 for the documented human A431, HeLa, or Jurkat lysate examples. Choose M03288 when its documented human, rat, or mouse sample types better match your experiment. Both have WB images; account for the reported band sizes when interpreting results.
Source: BosterBio PHF8 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q9UPP1.
- Human Protein Atlas. PHF8 tissue expression.
- PMC5436068 — target-verified WB comparison
- PMC7848440 — target-verified WB comparison
- PMC13137860 — target-verified WB comparison