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- Table of Contents
CDC25A / M-phase inducer phosphatase 1 · Western blot design guide
Design a Western Blot for CDC25A
Real validated CDC25A Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-CDC25A 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
CDC25A 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 | ~59.1 kDa | |
| Gel | 12–15% (standard starting point) | |
| Negative control | Suggested KO / knockdown lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated + Ubl conjugation | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated CDC25A Western Blot Protocols
The A01433-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | CT26 (40ug), PC12 (40ug), A549 (40ug) (catalog A01433-1) |
| Gel % | 12–15% (standard starting point) |
| Load | 20–30 µg total protein per lane; optimize for abundance (standard starting point) |
| Transfer | Standard semi-dry transfer; verify efficiency (standard starting point) |
| Membrane | 0.45 µm PVDF (standard starting point) |
| Blocking | 5% milk or 5% BSA in TBST (standard starting point) |
| Primary antibody | A01433-1; use the WB datasheet starting dilution (standard starting point) |
| 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 CDC25A Western Blot Band Size?
CDC25A has a predicted mass of 59.1 kDa; isoforms and phosphorylation could affect migration, but no empirical band or feature-specific shift is demonstrated.
| Band near 59.1 kDa | consistent with the predicted CDC25A size; confirm identity with controls |
| Two discrete bands | could reflect isoforms 1 and 2 if their migration differs; separation is unestablished |
| Doublet near 59.1 kDa | could reflect phosphorylation states; a visible shift is unestablished |
| Band migrating above 59.1 kDa | could reflect phosphorylation; identity and cause require controls |
💡Expected CDC25A appearanceUniProt predicts 59.1 kDa for CDC25A; no empirical band size is supplied, and the listed isoforms and phosphorylation sites do not establish a resolvable migration pattern, so confirm candidate bands with identity controls.
| UniProt predicted mass | sets a 59.1 kDa reference, not a measured band position |
| Isoform 1 | has no supplied isoform-specific mass or migration position |
| Isoform 2 | may differ from isoform 1, but its relative size is unspecified |
| Ser76 phosphorylation by CHEK1 | could affect migration, but no visible shift is demonstrated |
| Situation | Likely cause | Next action |
|---|---|---|
| Band higher than expected | phosphorylation is possible, but the cause of a shift is unestablished | compare phosphatase-treated and untreated samples and confirm band identity |
| Band lower than expected | the supplied features do not establish a smaller band position | check antibody specificity and compare samples with CDC25A depletion |
| Broad smear instead of sharp band | the supplied features do not establish a smear mechanism | check sample integrity and compare phosphatase-treated samples |
| Multiple bands | isoforms or phosphorylation states are possible, but resolved bands are unestablished | compare CDC25A-depleted samples and assess phosphatase sensitivity |
| Weak or no signal | CDC25A abundance or antibody detection may be insufficient | check loading and antibody performance with a positive control |
Experiment evidence
Western blot images showing CDC25A band
A01433-1 · CDC25A
A01433-2 · CDC25A
A01433-2 · CDC25A
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for CDC25A Western blot
🧪For positive controls for CDC25A in Western blot, you can use no HPA-supported positive sample because tissue and cell data were not supplied.
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.
⚠️Feasibility: HPA provides no positive or negative candidates, so sample controls cannot be selected from this evidence.
HPA tissue expression evidence for CDC25A
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 CDC25A Western Blot Tips
Deeper troubleshooting and optimisation questions for CDC25A, answered from its protein features.
How should CDC25A 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 CDC25A isoforms produce different bands?
Isoforms · UniProt lists two isoforms. Isoform 2 lacks canonical residues 144–183, a 40-residue segment, so it may migrate below isoform 1. The record gives no observed band positions; confirm isoform identity with an isoform-aware reagent or independent identification.
Isoform 2 lacks canonical residues 144–183, including the listed CHEK1 site at Ser178. Other listed coordinates refer to the supplied canonical sequence; downstream residue numbers may differ in isoform 2. Check the sequence and numbering convention used by any phosphosite-specific antibody.
Isoform 2 lacks canonical residues 144–183, including the listed CHEK1 site at Ser178. Other listed coordinates refer to the supplied canonical sequence; downstream residue numbers may differ in isoform 2. Check the sequence and numbering convention used by any phosphosite-specific antibody.
Which CDC25A phosphorylation sites matter when interpreting band patterns?
PTM · UniProt lists CHEK1 sites at Ser76, Ser124, Ser178, Ser279, Ser293, and Thr507; NEK11 sites at Ser79, Ser82, and Ser88; CHEK2 sites at Ser124, Ser279, and Ser293; and PLK3 sites at Ser513 and Ser519. Ser107 and Ser321 are also listed without an assigned enzyme. These features alone do not establish a visible mobility shift.
Does this guide establish induction of CDC25A?
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 CDC25A?
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 A01433-1 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 CDC25A bands be quantified?
Quantitation · Define the band or bands measured and use the same choice across samples. Two isoforms and 13 listed modified residues can complicate attribution of signal, while the record supplies no observed band pattern. If measuring a phosphosite, verify that the target residue exists in the isoform being measured.
Where should CDC25A migrate relative to its predicted mass?
Interpretation · The predicted mass is 59.1 kDa. No observed band position is supplied, so this record cannot establish a typical apparent mass or explain a difference from 59.1 kDa. Compare sample bands with a molecular weight marker and evaluate additional bands using the isoform and modification features below.
Consider isoform 2, which lacks canonical residues 144–183, alongside the listed phosphorylation sites. Neither feature alone identifies an observed band or proves a mobility shift. Check band position against the 59.1 kDa predicted mass, then validate identity with an independent reagent or identification method.
Consider isoform 2, which lacks canonical residues 144–183, alongside the listed phosphorylation sites. Neither feature alone identifies an observed band or proves a mobility shift. Check band position against the 59.1 kDa predicted mass, then validate identity with an independent reagent or identification method.
Boster reagents
CDC25A 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-M-phase inducer phosphatase 1 Cdc25A Antibody
Cat # A01433-1
Real WB data
Anti-Phospho-CDC25A(T507) Antibody
Cat # A01433-2
Two the supplier CDC25A antibodies have WB images: A01433-1 was tested on CT26, PC12, A549, L02, and HeLa lysates at 1:500; phospho-T507 antibody A01433-2 was tested on untreated and calyculin A-treated HeLa extracts. No publication evidence is supplied.
Which to pick: Choose A01433-1 for general CDC25A detection; it lists human, mouse, and rat reactivity and shows WB results across five cell lysates. Choose A01433-2 when measuring T507 phosphorylation in human samples; its shown WB context is HeLa cells with and without calyculin A.
Source: BosterBio CDC25A gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P30304.
- Human Protein Atlas. CDC25A tissue expression.
- PMC5116667 — target-verified WB comparison
- PMC8284019 — target-verified WB comparison
- PMC5149581 — target-verified WB comparison