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CDKN1A / Cyclin-dependent kinase inhibitor 1 · Western blot design guide
Design a Western Blot for CDKN1A
Real validated CDKN1A Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-CDKN1A 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
CDKN1A 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 | ~18.1 kDa | |
| Observed band | ~21 kDa | |
| Gel | 5–20% (catalog A00145-1) | |
| Positive control | Cervix (IHC candidate; verify WB) +4 more | |
| Negative control | Caudate (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 | 1 isoform(s) |
Section 1
Real Curated CDKN1A Western Blot Protocols
The A00145-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human Hela, human HepG2, human MCF-7 (catalog A00145-1) |
| Gel % | 5–20% (catalog A00145-1) |
| Load | 30 ug; reducing conditions (catalog A00145-1) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A00145-1) |
| Membrane | nitrocellulose membrane (catalog A00145-1) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A00145-1) |
| Primary antibody | A00145-1 · 0.5 μg/mL (catalog A00145-1) |
| Primary incubation | overnight at 4°C (catalog A00145-1) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A00145-1) |
| Secondary incubation | 1.5 hour at RT (catalog A00145-1) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A00145-1) |
| Detection | ECL (catalog A00145-1) |
Section 2
What Is the Expected CDKN1A Western Blot Band Size?
CDKN1A is predicted at 18.1 kDa and observed at ~21 kDa; the cause of the difference is not established.
| Band at ~21 kDa | Empirical CDKN1A band; confirm identity with appropriate controls |
| Band near 18.1 kDa | Near the UniProt predicted mass; identity requires confirmation |
| Close bands near the main band | Possible modification states, including phosphorylation; separation is not established |
| Little or no band in a lysate | CDKN1A signal may be below detection in that sample; verify expression and assay performance |
💡Expected CDKN1A appearanceUniProt predicts 18.1 kDa, while antibody QC detects a ~21 kDa band in whole-cell lysates; the cause of this difference is unestablished, so confirm band identity with controls.
| UniProt predicted mass | 18.1 kDa from sequence, compared with the empirical ~21 kDa band |
| Phosphorylation at Thr80, Ser114, Ser130, Thr145, and Ser146 | May alter migration if present; no visible shift is established |
| N-terminal acetylation at Ser2 | Is documented, but its effect on apparent band size is unestablished |
| In vitro phosphorylation at Ser160 | Is documented in vitro; an endogenous band shift is unestablished |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | CDKN1A abundance may be below detection in the sampled cells | Check expression in the sample and include a known positive lysate |
| Band higher than expected | The documented ~21 kDa band exceeds the 18.1 kDa sequence prediction for an undetermined reason | Compare with the QC band and verify identity by CDKN1A depletion |
| Band lower than expected | Identity or sample integrity is uncertain; no cleavage feature is supplied | Check loading, sample integrity, and CDKN1A depletion |
| Multiple bands | Modification states are possible, but distinct migrating forms are unproven | Use CDKN1A depletion to identify specific bands |
| Weak or no signal | Low protein abundance or inadequate antibody detection | Include a positive lysate and check transfer and antibody conditions |
| Fragments below expected size | Sample degradation is possible; no defined cleavage product is supplied | Check sample handling and test whether the fragments disappear with CDKN1A depletion |
Experiment evidence
Western blot images showing CDKN1A band
A00145-1 · CDKN1A
M00145-3 · CDKN1A
M00145-2 · CDKN1A
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for CDKN1A Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for CDKN1A in Western blot, you can use cervix tissue, which HPA rates as highly expressed.
Positive control: Cervix (IHC candidate; verify WB)
Negative control: Caudate (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside the samples.
⚠️Feasibility: HPA lists caudate as not detected, making it a candidate negative tissue control.
HPA tissue expression evidence for CDKN1A
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 |
|---|---|---|---|---|
| Cervix | squamous epithelial cells | High | Protein (IHC) | HPA → |
| Placenta | cytotrophoblasts | High | Protein (IHC) | HPA → |
| Skin | endothelial cells | High | Protein (IHC) | HPA → |
| Vagina | squamous epithelial cells | High | Protein (IHC) | HPA → |
| Adipose tissue | adipocytes | Medium | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Caudate | glial cells | Not detected | Protein (IHC) | HPA → |
| Cerebellum | cells in granular layer | Not detected | Protein (IHC) | HPA → |
| Cerebral cortex | endothelial cells | Not detected | Protein (IHC) | HPA → |
| Heart muscle | cardiomyocytes | Not detected | Protein (IHC) | HPA → |
| Hippocampus | glial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CDKN1A Western Blot Tips
Deeper troubleshooting and optimisation questions for CDKN1A, answered from its protein features.
How should CDKN1A 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 CDKN1A isoforms explain multiple bands?
Isoforms · The supplied record lists one isoform and no alternative sequence. It therefore does not support assigning additional bands to splice isoforms. Check whether extra bands reproduce across samples before interpreting them as CDKN1A.
Which CDKN1A phosphorylation sites matter when interpreting bands?
PTM · UniProt lists Thr80 by LKB1, Ser114 by GSK3-beta, Ser130, Thr145 by PKA, PKB/AKT1, PIM1 and PIM2, Ser146 by PKC and NUAK1, and Ser160 by PKC in vitro. It also lists N-acetylserine at position 2. These are UniProt coordinates; antibody or paper numbering may differ. None alone establishes a resolvable band shift.
Quantify the approximately 21 kDa band consistently across matched samples. If using a site-specific antibody, check that its stated residue matches the UniProt coordinate and compare its signal with total CDKN1A. A change in the site-specific signal alone does not establish a change in total protein.
The supplied record lists no glycosylation sites, signal peptide, or propeptide, so those features provide no basis for assigning an extra band. The listed phosphorylation and acetylation sites also do not prove that any extra band is modified CDKN1A. Compare reproducibility and antibody specificity before assigning it.
Quantify the approximately 21 kDa band consistently across matched samples. If using a site-specific antibody, check that its stated residue matches the UniProt coordinate and compare its signal with total CDKN1A. A change in the site-specific signal alone does not establish a change in total protein.
The supplied record lists no glycosylation sites, signal peptide, or propeptide, so those features provide no basis for assigning an extra band. The listed phosphorylation and acetylation sites also do not prove that any extra band is modified CDKN1A. Compare reproducibility and antibody specificity before assigning it.
Does this guide establish induction of CDKN1A?
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 CDKN1A Western blot?
Transfer · CDKN1A is a 164-residue protein with a predicted mass of 18.1 kDa and an observed band near 21 kDa. Choose a transfer setup that retains proteins in this size range, then verify transfer using a marker or total-protein stain. The supplied features do not specify a particular membrane or transfer procedure.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A00145-1 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 CDKN1A 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 CDKN1A appear near 21 kDa instead of 18.1 kDa?
Interpretation · The supplied observed band is approximately 21 kDa, while the sequence predicts 18.1 kDa. The listed modifications do not establish the cause of that difference or prove a visible shift. Use a molecular weight marker and compare the band across matched samples before assigning it to CDKN1A.
The supplied record places CDKN1A in both nucleus and cytoplasm. Either fraction may contain a CDKN1A band, so compare matched fractions with appropriate fraction controls. Location alone does not establish how much protein should appear in either fraction.
The supplied record places CDKN1A in both nucleus and cytoplasm. Either fraction may contain a CDKN1A band, so compare matched fractions with appropriate fraction controls. Location alone does not establish how much protein should appear in either fraction.
Boster reagents
CDKN1A 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-P21/CDKN1A Antibody Picoband®
Cat # A00145-1
Real WB data
Anti-p21 CDKN1A Rabbit Monoclonal Antibody
Cat # M00145-3
Real WB data
Anti-p21 CDKN1A Rabbit Monoclonal Antibody
Cat # M00145-2
Three the supplier anti-CDKN1A antibodies have WB images. A00145-1 shows an approximately 21 kDa band in five human cell lysates with reported blot conditions. The monoclonal images show cell lysate results but provide fewer experimental details; no independent validation is supplied.
Which to pick: For human samples, A00145-1 has the most detailed WB caption and a reported 21 kDa band. M00145-3 is another human option. M00145-2 lists human, mouse and rat reactivity, although its pictured WB lanes use human cell lysates.
Source: BosterBio CDKN1A gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P38936.
- Human Protein Atlas. CDKN1A tissue expression.
- PMC11373099 — target-verified WB comparison
- PMC4742851 — target-verified WB comparison
- PMC7993694 — target-verified WB comparison