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- Table of Contents
TNIK / TRAF2 and NCK-interacting protein kinase · Western blot design guide
Design a Western Blot for TNIK
Source-linked TNIK Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-TNIK 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
TNIK 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 | ~154.9 kDa | |
| Observed band | ~180 and 150 kDa | |
| Gel | 5–20% (catalog A03934-3) | |
| Positive control | Appendix (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 | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 8 isoform(s) |
Section 1
Source-Linked TNIK Western Blot Protocol Options
The A03934-3 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human K562, human HEL (catalog A03934-3) |
| Gel % | 5–20% (catalog A03934-3) |
| Load | 30 ug; reducing conditions (catalog A03934-3) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A03934-3) |
| Membrane | nitrocellulose membrane (catalog A03934-3) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A03934-3) |
| Primary antibody | A03934-3 · 0.5 μg/mL (catalog A03934-3) |
| Primary incubation | overnight at 4°C (catalog A03934-3) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A03934-3) |
| Secondary incubation | 1.5 hour at RT (catalog A03934-3) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A03934-3) |
| Detection | ECL (catalog A03934-3) |
Section 2
What Is the Expected TNIK Western Blot Band Size?
TNIK is predicted at 154.9 kDa, while antibody QC shows bands near 180 and 150 kDa; the cause of their difference is not established.
| Band near 150 kDa | Empirical TNIK-reactive band close to the 154.9 kDa predicted mass |
| Band near 180 kDa | Empirical TNIK-reactive band whose higher migration position is unexplained |
| Bands near 180 and 150 kDa | Both bands were observed in reducing whole-cell lysates; their distinct identities are unresolved |
| Band near 155 kDa | Position expected from the UniProt predicted mass, subject to band-identity confirmation |
| Weak band in a soluble fraction | TNIK may also reside in nuclear, endosomal, or cytoskeletal fractions |
💡Expected TNIK appearanceTNIK has a predicted mass of 154.9 kDa, while antibody QC reports bands near 180 and 150 kDa; their identities and the cause of the difference require ordinary band-identity controls.
| UniProt predicted mass | 154.9 kDa is the sequence-based reference; empirical TNIK-reactive bands occur near 180 and 150 kDa |
| Splice isoform 1 | Its separate apparent size is not supplied |
| Splice isoforms 2, 3, and 4 | Sequence differences may affect apparent size, but their relative sizes are not supplied |
| Splice isoforms 5, 6, 7, and 8 | Sequence differences may affect apparent size, but their relative sizes are not supplied |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | TNIK distribution across nuclear, endosomal, and cytoskeletal compartments may limit recovery in the prepared lysate | Check extraction and relevant fractions with a positive control |
| Band higher than expected | The observed 180 kDa band exceeds the 154.9 kDa prediction; its cause is not established | Confirm identity by TNIK depletion and compare with the reported QC pattern |
| Band lower than expected | The observed 150 kDa band lies near the predicted mass; its precise identity is unconfirmed | Compare with the reported QC pattern and confirm by TNIK depletion |
| Multiple bands | Isoforms 1 through 8 exist, but their migration and contribution to the observed bands are unknown | Confirm band identities by TNIK depletion and assess expressed isoforms if needed |
| Weak or no signal | TNIK may be poorly recovered from the sampled cellular fraction | Check nuclear and cytoskeletal fractions alongside a positive control |
| Fragments below expected size | Fragment identity is not established by the supplied features | Check sample integrity and confirm TNIK specificity by depletion |
Experiment evidence
Western blot images showing TNIK band
A03934-3 · TNIK
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for TNIK Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for TNIK in Western blot, you can use appendix tissue lysate, where HPA reports high expression.
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 such as Ponceau alongside the samples.
⚠️Feasibility: HPA identifies high-expression and not-detected tissues, making a tissue control pair feasible.
HPA tissue expression evidence for TNIK
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 | glandular cells | High | Protein (IHC) | HPA → |
| Colon | glandular cells | High | Protein (IHC) | HPA → |
| Duodenum | glandular cells | High | Protein (IHC) | HPA → |
| Gallbladder | glandular cells | High | Protein (IHC) | HPA → |
| Rectum | glandular cells | High | 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 → |
| Cervix | glandular cells | Not detected | Protein (IHC) | HPA → |
| Esophagus | squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Ovary | ovarian stroma cells | Not detected | Protein (IHC) | HPA → |
| Skin | fibroblasts | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced TNIK Western Blot Tips
Deeper troubleshooting and optimisation questions for TNIK, answered from its protein features.
How should TNIK 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 TNIK isoforms produce different bands?
Isoforms · Eight isoforms are listed. Relative to the canonical sequence, residues 445–473 are missing in isoforms 2, 5, 6, and 8; 537–591 in 3, 5, 7, and 8; and 795–802 in 4, 6, 7, and 8. Check whether the antibody epitope falls within a deleted segment when comparing bands.
Isoforms 3, 5, 7, and 8 lack canonical residues 537–591, which include the listed S560, S570, and T581 sites. Account for that deletion when interpreting site-specific signals. Coordinates here follow the supplied canonical UniProt sequence.
Isoforms 3, 5, 7, and 8 lack canonical residues 537–591, which include the listed S560, S570, and T581 sites. Account for that deletion when interpreting site-specific signals. Coordinates here follow the supplied canonical UniProt sequence.
Which TNIK phosphorylation sites matter when interpreting band changes?
PTM · UniProt lists phosphothreonine at T187 and T581, and phosphoserine at S324, S326, S560, S570, S600, S608, S610, S640, S678, S680, S688, S701, S707, S720, S764, S766, S769, and S959. These are canonical UniProt coordinates; antibody or paper numbering may differ. Their presence alone does not establish a visible mobility shift.
Does this guide establish induction of TNIK?
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 TNIK Western blot?
Transfer · The supplied features give a predicted mass of 154.9 kDa but do not specify a transfer method. Choose and verify a transfer setup that recovers proteins around the observed ~150 and ~180 kDa positions; check the membrane and remaining gel before interpreting a missing band.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A03934-3 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 TNIK bands be quantified across samples?
Quantitation · Quantify the same verified band across samples and report ~150 and ~180 kDa bands separately until their identities are established. TNIK has eight isoforms and is listed in the nucleus, cytoplasm, recycling endosomes, and cytoskeletal fraction, so keep sample preparation and fraction choice consistent.
Do the ~150 and ~180 kDa bands match TNIK's predicted mass?
Interpretation · TNIK's predicted mass is 154.9 kDa, close to the observed ~150 kDa band. The ~180 kDa band has a higher apparent mass. TNIK's listed phosphorylation sites and splice variants do not, by themselves, establish why either band migrates there. Verify band identity before assigning either band to a specific form.
First compare each band's apparent mass with the 154.9 kDa prediction and the observed ~150 and ~180 kDa bands. Consider the documented splice deletions and antibody epitope coverage. Listed phosphorylation sites are candidates for investigation, but neither a band shift nor its cause follows from site annotation alone.
First compare each band's apparent mass with the 154.9 kDa prediction and the observed ~150 and ~180 kDa bands. Consider the documented splice deletions and antibody epitope coverage. Listed phosphorylation sites are candidates for investigation, but neither a band shift nor its cause follows from site annotation alone.
Boster reagents
TNIK 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-TNIK Antibody Picoband®
Cat # A03934-3
The catalog reports one anti-TNIK antibody, A03934-3, with stated human and mouse reactivity. Its Western blot image uses human K562 and HEL whole-cell lysates and reports bands near 180 and 150 kDa, versus an expected 155 kDa. No publication or orthogonal validation evidence is supplied.
Which to pick: A03934-3 is the only listed option. Its image provides a starting reference for human K562 and HEL lysates at 0.5 μg/mL antibody. Mouse reactivity is listed, but the supplied image does not test a mouse sample.
Source: BosterBio TNIK gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q9UKE5.
- Human Protein Atlas. TNIK tissue expression.
- PMC12058923 — target-verified WB comparison
- PMC3105225 — target-verified WB comparison