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PLK4 / Serine/threonine-protein kinase PLK4 · Western blot design guide
Design a Western Blot for PLK4
Real validated PLK4 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-PLK4 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
PLK4 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 | ~109 kDa | |
| Gel | 8–10% (standard starting point) | |
| Positive control | Testis (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 + Acetylated | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 3 isoform(s) |
Section 1
Real Curated PLK4 Western Blot Protocols
The A01947 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 A01947) |
| Gel % | 8–10% (standard starting point) |
| Load | 20–30 µg total protein per lane; optimize for abundance (standard starting point) |
| Transfer | Wet/tank transfer; optimize duration (standard starting point) |
| Membrane | 0.45 µm PVDF (standard starting point) |
| Blocking | Blocking buffer: 3% nonfat dry milk in TBST (catalog A01947) |
| Primary antibody | A01947 · 1:1000 (catalog A01947) |
| Primary incubation | Overnight at 4 °C (standard starting point) |
| Secondary antibody | Goat Anti-Rabbit IgG, 1:10000 (catalog A01947) |
| Secondary incubation | 1 h at room temperature (standard starting point) |
| Wash | 3 × 5 min in TBST (standard starting point) |
| Detection | ECL (catalog A01947) |
Section 2
What Is the Expected PLK4 Western Blot Band Size?
PLK4 has a predicted 109 kDa monomer; homodimerization and isoforms could affect bands, but their migration effects are unverified and no empirical band is supplied.
| Band near 109 kDa | consistent with the predicted PLK4 monomer size; migration is unverified |
| Band near 218 kDa under nonreducing conditions | could reflect the reported homodimer |
| Several bands at different sizes | could reflect isoforms 1, 2, and 3; distinct migration is unverified |
| Band in a centrosome-enriched fraction | consistent with PLK4 localization at the centrosome |
💡Expected PLK4 appearanceUniProt predicts a 109 kDa PLK4 monomer, but no empirical band size is supplied; confirm any candidate band with an appropriate PLK4 identity control.
| Predicted monomer mass | sets a 109 kDa reference, not a measured migration position |
| Homodimer by similarity | could yield a band near twice the monomer size under nonreducing or incompletely reducing conditions |
| Isoforms 1, 2, and 3 | could differ in size, but their masses and migration differences are not supplied |
| Phosphoserines at residues 401, 665, and 817 | are documented modifications without a demonstrated band shift |
| N6-acetyllysines at residues 45 and 46 | are documented modifications without a demonstrated band shift |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | PLK4 localization at centrioles may limit its representation in whole-cell lysate | check a centrosome-enriched fraction and a PLK4-positive control |
| Band higher than expected | reported homodimer may persist with incomplete reduction | compare reducing and nonreducing preparations and verify band identity |
| Band lower than expected | an alternative isoform is possible, but its migration is unknown | check antibody epitope coverage and verify band identity |
| Multiple bands | isoforms 1, 2, and 3 are annotated, but distinct bands are unverified | compare isoform-aware controls and verify each band's identity |
| Weak or no signal | PLK4 may be poorly represented in the sampled fraction | check a centrosome-enriched fraction and a PLK4-positive control |
Experiment evidence
Western blot images showing PLK4 band
A01947 · PLK4
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for PLK4 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for PLK4 in Western blot, you can use testis lysate, the highest-scoring HPA positive tissue.
Positive control: Testis (IHC candidate; verify WB)
Negative control: Adipose tissue (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside the samples.
⚠️Feasibility: A tissue negative control is available, although low PLK4 signal may still be detectable by Western blot.
HPA tissue expression evidence for PLK4
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 |
|---|---|---|---|---|
| Testis | elongated or late spermatids | High | Protein (IHC) | HPA → |
| Appendix | glandular cells | Medium | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Medium | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | Medium | Protein (IHC) | HPA → |
| Cervix | glandular cells | Medium | 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 | Not detected | Protein (IHC) | HPA → |
| Breast | adipocytes | Not detected | Protein (IHC) | HPA → |
| Caudate | glial cells | Not detected | Protein (IHC) | HPA → |
| Cerebellum | cells in granular layer | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced PLK4 Western Blot Tips
Deeper troubleshooting and optimisation questions for PLK4, answered from its protein features.
How should PLK4 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.
How could PLK4 isoforms affect band interpretation?
Isoforms · Isoform 3 lacks canonical residues 1–41, and isoform 2 lacks residues 43–74. Either deletion could produce a smaller protein. Check which region the antibody recognizes before assigning a band to an isoform.
Which PLK4 modifications matter when comparing bands?
PTM · UniProt lists N6-acetyllysine at canonical positions 45 and 46 and phosphoserine at 401, 665, and 817. Isoform 2 lacks the segment containing canonical positions 45 and 46. State the sequence convention when comparing site numbers; modification-site listings do not establish a detectable band shift.
Does this guide establish induction of PLK4?
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 PLK4 Western blot?
Transfer · PLK4 has a predicted mass of 109 kDa. Use transfer conditions suitable for a protein around that size and verify transfer near 109 kDa with a molecular-weight marker. The supplied features do not identify a specific transfer method.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A01947 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 PLK4 bands be quantified?
Quantitation · Quantify comparable bands from the same sample fraction and specify which isoform or band was measured. PLK4 is listed at the centrosome and in the nucleus, so fraction choice can affect what is detected. The features provide no basis for assuming that every band represents the same PLK4 form.
Why might a PLK4 band differ from its predicted mass?
Interpretation · The supplied predicted mass is 109 kDa, but no observed band is available for comparison. PLK4 has three isoforms and listed acetylation and phosphorylation sites. These features alone cannot establish a visible shift or explain a particular apparent mass.
Compare them with the 109 kDa prediction and the deletions in isoforms 2 and 3, then check whether the antibody recognizes the retained sequence. The listed modifications are possible considerations, but the supplied features cannot identify an unexpected band on their own.
Compare them with the 109 kDa prediction and the deletions in isoforms 2 and 3, then check whether the antibody recognizes the retained sequence. The listed modifications are possible considerations, but the supplied features cannot identify an unexpected band on their own.
Boster reagents
PLK4 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-PLK4 Antibody
Cat # A01947
the supplier A01947 is an anti-PLK4 antibody listed for human, mouse and rat. Its Western blot image uses cell-line extracts at 25 µg per lane and a 1:1000 primary dilution; the supplied caption does not identify the cell lines or document species-specific validation.
Which to pick: A01947 is the only listed option and has a Western blot image. Use the reported 1:1000 primary dilution as a starting point, and confirm performance in your own sample.
Source: BosterBio PLK4 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry O00444.
- Human Protein Atlas. PLK4 tissue expression.
- PMC12327949 — target-verified WB comparison
- PMC12374821 — target-verified WB comparison
- PMC9251049 — target-verified WB comparison