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NEK2 / Serine/threonine-protein kinase Nek2 · Western blot design guide
Design a Western Blot for NEK2
Real validated NEK2 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-NEK2 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
NEK2 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 | ~51.8 kDa | |
| Gel | 12–15% (standard starting point) | |
| Positive control | Appendix (IHC candidate; verify WB) +3 more | |
| Negative control | Adipose tissue (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 Hallmark membership | |
| Isoform | 4 isoform(s) |
Section 1
Real Curated NEK2 Western Blot Protocols
The M01606 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | 293T cell lysate (catalog M01606) |
| 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 | M01606; 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 NEK2 Western Blot Band Size?
NEK2 is predicted at 51.8 kDa; isoforms, phosphorylation and dimerization are possible band modifiers, but their effects on migration have not been demonstrated here.
| Band near 51.8 kDa | consistent with the predicted NEK2 mass; confirm its identity with antibody controls |
| More than one band near the expected region | could reflect isoforms 1, 2, 3 or 4, or different phosphorylation states; distinct migration is unproven |
| Band with altered mobility | may reflect NEK2 phosphorylation, but a visible shift is unproven |
| Band near twice the monomer size | could reflect an NEK2 dimer only if it survives sample preparation |
💡Expected NEK2 appearanceNEK2 has a predicted mass of 51.8 kDa, but no empirical band size is supplied; use antibody specificity controls to identify any band and assess possible isoform or phosphorylation effects.
| Predicted NEK2 mass | 51.8 kDa is the sequence-based reference, not a measured migration |
| Isoforms 1 and 2 | alternative splicing could change apparent size; their individual masses are unknown |
| Isoforms 3 and 4 | alternative splicing could change apparent size; their individual masses are unknown |
| NEK2 homo- and heterodimers | could yield a higher band if a dimer survives sample preparation; no covalent linkage is listed |
| Situation | Likely cause | Next action |
|---|---|---|
| Band higher than expected | a NEK2 dimer may have survived sample preparation | compare denaturing conditions and confirm band identity with a second antibody |
| Band lower than expected | an alternative isoform is possible, but isoform masses are unavailable | check which isoforms the antibody recognizes and compare with an independent antibody |
| Multiple bands | alternative isoforms or phosphorylation states may differ in migration | compare isoform coverage and use phosphatase treatment with appropriate controls |
| Weak or no signal | NEK2 may be underrepresented in the sampled cellular fraction | check loading and antibody performance, then compare nuclear and cytoskeletal fractions |
| Fragments below expected size | fragment identity is not established by the supplied features | check sample integrity and confirm NEK2 identity with an independent antibody |
Experiment evidence
Western blot images showing NEK2 band
M01606 · NEK2
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for NEK2 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for NEK2 in Western blot, you can use appendix tissue lysate.
Positive control: Appendix (IHC candidate; verify WB)
Negative control: Adipose tissue (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: Appendix has only medium HPA signal, so the positive band may be modest.
HPA tissue expression evidence for NEK2
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 |
|---|---|---|---|---|
| Appendix | lymphoid tissue | Medium | Protein (IHC) | HPA → |
| Lymph node | germinal center cells | Medium | Protein (IHC) | HPA → |
| Testis | pachytene spermatocytes | Medium | Protein (IHC) | HPA → |
| Tonsil | germinal center 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 → |
| Bone marrow | hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | adipocytes | Not detected | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced NEK2 Western Blot Tips
Deeper troubleshooting and optimisation questions for NEK2, answered from its protein features.
How should NEK2 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 NEK2 isoforms produce bands of different sizes?
Isoforms · Yes. Four isoforms are listed. Relative to the canonical sequence, isoform 2 lacks residues 385–445, isoform 3 lacks 327–445, and isoform 4 lacks 371–378. Isoforms 2 and 3 also have sequence replacements. Check which isoforms the antibody recognizes before assigning bands.
Map the antibody's target to each isoform before interpreting a negative signal. Isoform 3 lacks canonical residues 327–445, including the listed STK3/MST2 sites. Isoform 2 lacks 385–445, while isoform 4 lacks 371–378. Use UniProt canonical coordinates when comparing site labels.
Map the antibody's target to each isoform before interpreting a negative signal. Isoform 3 lacks canonical residues 327–445, including the listed STK3/MST2 sites. Isoform 2 lacks 385–445, while isoform 4 lacks 371–378. Use UniProt canonical coordinates when comparing site labels.
Which phosphorylation sites matter when interpreting NEK2 bands?
PTM · UniProt lists 17 modified residues. Autophosphorylation is annotated at Thr170, Ser171, Thr175, Thr179, and Ser241; STK3/MST2 phosphorylates Ser356, Ser365, Ser406, and Ser438. These are UniProt canonical-sequence coordinates; antibody or paper numbering may differ. Their presence does not establish a visible mobility shift.
Does this guide establish induction of NEK2?
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 NEK2?
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 M01606 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 NEK2 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.
Should NEK2 migrate at its predicted 51.8 kDa?
Interpretation · 51.8 kDa is the predicted mass of the 445-residue canonical sequence. No observed band position is supplied. Use it as a reference, but do not infer a band shift or its cause from the listed phosphorylation sites alone.
Keep the detected isoform and sample fraction consistent. NEK2 is associated with the cell cycle, and isoform 1 is listed in nuclear and cytoplasmic locations, including centrosomes and spindle poles. A change in signal from one fraction alone need not represent a change in total NEK2.
First check whether the antibody recognizes isoforms 1–4 and whether shorter isoform sequences fit the result. NEK2 is also annotated as phosphorylated, but the listed sites alone cannot explain a particular band position. An unexpected band cannot be assigned from these features without additional evidence.
Isoforms 1, 2, and 4 are annotated to form homo- and heterodimers. That interaction does not establish that a dimer survives sample preparation or appears as a separate band. Check the assay conditions and isoform recognition before assigning a higher band to a dimer.
Keep the detected isoform and sample fraction consistent. NEK2 is associated with the cell cycle, and isoform 1 is listed in nuclear and cytoplasmic locations, including centrosomes and spindle poles. A change in signal from one fraction alone need not represent a change in total NEK2.
First check whether the antibody recognizes isoforms 1–4 and whether shorter isoform sequences fit the result. NEK2 is also annotated as phosphorylated, but the listed sites alone cannot explain a particular band position. An unexpected band cannot be assigned from these features without additional evidence.
Isoforms 1, 2, and 4 are annotated to form homo- and heterodimers. That interaction does not establish that a dimer survives sample preparation or appears as a separate band. Check the assay conditions and isoform recognition before assigning a higher band to a dimer.
Boster reagents
NEK2 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-NEK2 Rabbit Monoclonal Antibody
Cat # M01606
The catalog reports a rabbit monoclonal anti-NEK2 antibody with Human reactivity. Its Western blot image shows NEK2 expression in 293T cell lysate. No publication evidence or additional sample validation is supplied.
Which to pick: M01606 is the only listed option. Choose it for a Human NEK2 Western blot when the reported 293T cell lysate image is relevant to your experiment; performance in other samples is not documented here.
Source: BosterBio NEK2 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P51955.
- Human Protein Atlas. NEK2 tissue expression.
- PMC10932871 — target-verified WB comparison
- PMC5494456 — target-verified WB comparison
- PMC5802024 — target-verified WB comparison