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NUAK1 / NUAK family SNF1-like kinase 1 · Western blot design guide
Design a Western Blot for NUAK1
Source-linked NUAK1 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-NUAK1 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
NUAK1 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 | ~74.3 kDa | |
| Observed band | ~74 kDa | |
| Gel | 10% (catalog A03575-1) | |
| Negative control | Suggested KO / knockdown lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated + Acetylated | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 2 isoform(s) |
Section 1
Source-Linked NUAK1 Western Blot Protocol Options
The A03575-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human U251, human A549, human Hela, human RT4, rat brain, rat heart, mouse brain, mouse heart (catalog A03575-1) |
| Gel % | 10% (catalog A03575-1) |
| Load | 30 ug; reducing conditions (catalog A03575-1) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A03575-1) |
| Membrane | nitrocellulose membrane (catalog A03575-1) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A03575-1) |
| Primary antibody | A03575-1 · 1:500 (catalog A03575-1) |
| Primary incubation | overnight at 4°C (catalog A03575-1) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:1000 (catalog A03575-1) |
| Secondary incubation | 1.5 hour at RT (catalog A03575-1) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A03575-1) |
| Detection | ECL (catalog A03575-1) |
Section 2
What Is the Expected NUAK1 Western Blot Band Size?
NUAK1 is predicted at 74.3 kDa and observed at approximately 74 kDa; documented isoforms have no supplied individual band sizes.
| Band at approximately 74 kDa | Matches the observed NUAK1 band and its 74.3 kDa predicted mass |
| Several bands near 74 kDa | Isoforms 1 and 2 are possible contributors, but distinct migration is unconfirmed |
| Slightly shifted band near 74 kDa | Phosphorylation could affect migration, but a visible shift is unconfirmed |
| Bands in nuclear and cytoplasmic fractions | Consistent with NUAK1 localization in both compartments |
💡Expected NUAK1 appearanceNUAK1 has a predicted mass of 74.3 kDa and an observed band at approximately 74 kDa; confirm band identity with a positive control and NUAK1 depletion.
| Predicted NUAK1 mass | 74.3 kDa predicted; the observed band is approximately 74 kDa |
| Splice isoform 1 | Its individual mass and migration relative to isoform 2 are not supplied |
| Splice isoform 2 | Its individual mass and migration relative to isoform 1 are not supplied |
| Alternative splicing into isoforms 1 and 2 | May alter apparent size, but distinct bands are unconfirmed |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | NUAK1 may be poorly recovered from nuclear or cytoplasmic material | Check lysate preparation and compare with a positive control lysate |
| Band higher than expected | An isoform or altered migration is possible; the cause is unestablished | Compare with the approximately 74 kDa control band and test NUAK1 depletion |
| Band lower than expected | An isoform or degradation is possible; the cause is unestablished | Compare fresh lysate with a positive control and test NUAK1 depletion |
| Multiple bands | Isoforms 1 and 2 are documented, but distinct band sizes are unconfirmed | Use NUAK1 depletion to identify specific bands |
| Weak or no signal | NUAK1 recovery or abundance may be insufficient | Check loading and recovery with a positive control lysate |
Experiment evidence
Western blot images showing NUAK1 band
A03575-1 · NUAK1
A03575 · NUAK1
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for NUAK1 Western blot
🧪For positive controls for NUAK1 in Western blot, you can use a validated positive sample, but the supplied HPA data identifies none.
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 the samples.
⚠️Feasibility: HPA provides no expression data, so a tissue-based positive control cannot be selected.
HPA tissue expression evidence for NUAK1
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 NUAK1 Western Blot Tips
Deeper troubleshooting and optimisation questions for NUAK1, answered from its protein features.
How should NUAK1 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 NUAK1 isoforms produce different bands?
Isoforms · Yes. Isoform 2 replaces canonical residues 182–208 and lacks residues 209–661, leaving it much shorter than the 661-residue canonical isoform. Its apparent band position is not supplied; check whether the antibody recognizes a region retained in isoform 2.
Isoform 2 retains canonical Ser22 but lacks the canonical region containing Thr211, Ser455, and Ser600. A signal from an antibody against one of those latter sites cannot be assigned to isoform 2 from these features.
Isoform 2 retains canonical Ser22 but lacks the canonical region containing Thr211, Ser455, and Ser600. A signal from an antibody against one of those latter sites cannot be assigned to isoform 2 from these features.
Which phosphorylation sites are annotated for NUAK1?
PTM · UniProt lists Ser22, Thr211 phosphorylated by LKB1, Ser455, and Ser600 phosphorylated by PKB/AKT1. These are canonical UniProt coordinates; antibody or paper numbering may differ. Site presence alone does not predict a visible band shift.
For a phosphosite-specific blot, compare the phosphosite signal with total NUAK1 in the same samples. UniProt assigns Thr211 phosphorylation to LKB1 and Ser600 phosphorylation to PKB/AKT1, but the supplied features do not establish how any treatment changes either signal.
For a phosphosite-specific blot, compare the phosphosite signal with total NUAK1 in the same samples. UniProt assigns Thr211 phosphorylation to LKB1 and Ser600 phosphorylation to PKB/AKT1, but the supplied features do not establish how any treatment changes either signal.
Does this guide establish induction of NUAK1?
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 NUAK1?
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 A03575-1 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 NUAK1 bands be quantified across fractions?
Quantitation · NUAK1 is reported in both nucleus and cytoplasm, so specify which fraction each measurement represents. Use consistent loading and a total-NUAK1 signal when comparing phosphosite signals. Check antibody recognition of both isoforms before treating all bands as total NUAK1.
Why does NUAK1 appear near 74 kDa?
Interpretation · The observed band near 74 kDa agrees with the predicted 74.3 kDa mass of canonical NUAK1. The listed modifications do not establish a visible shift or explain a difference between calculated and apparent mass.
Consider the shorter isoform 2 and antibody epitope coverage when assessing a lower band. UniProt lists no signal peptide, propeptide, or glycosylation sites here. Its annotated phosphorylation and N-terminal acetylation do not, by themselves, establish the cause of an additional band.
Consider the shorter isoform 2 and antibody epitope coverage when assessing a lower band. UniProt lists no signal peptide, propeptide, or glycosylation sites here. Its annotated phosphorylation and N-terminal acetylation do not, by themselves, establish the cause of an additional band.
Boster reagents
NUAK1 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-ARK5/NUAK1 antibody
Cat # A03575-1
Real WB data
Anti-NUAK family SNF1-like kinase 1 NUAK1 Antibody
Cat # A03575
Two the supplier anti-NUAK1 antibodies have Western blot images. A03575-1 shows an approximately 74 kDa band in the listed human cell, rat tissue, and mouse tissue lysates. A03575 has a brief DU145 lysate caption; it provides less detail about the result.
Which to pick: Choose A03575-1 for rat samples or when a documented band and detailed blot conditions are useful. Both list human and mouse reactivity. A03575 shows a DU145 lysate blot at 1:1000, but its caption gives limited validation detail.
Source: BosterBio NUAK1 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry O60285.
- Human Protein Atlas. NUAK1 tissue expression.
- PMC7280971 — target-verified WB comparison
- PMC10621130 — target-verified WB comparison
- PMC11789290 — target-verified WB comparison