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ITK · Western blot design guide
Design a Western Blot for ITK
Real validated ITK Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-ITK WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Last reviewed: May 2026 · Scientific review: Boster Bio technical team
ITK 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 | ~71.8 kDa | |
| Observed band | Approximately 72 kDa | |
| Gel | 10–12% | |
| Negative control | siRNA / KO lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated + Ubiquitinated | |
| Caveat | Phosphorylation-induced mobility shift | |
| Regulation | Allograft rejection | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated ITK Western Blot Protocols
Literature-validated Western blot parameters for ITK — gel percentage, transfer, blocking, antibody incubation and detection, extracted from published methods.
| Sample / lysate | human Jurkat , Lane 2: human MOLT-4 . After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-ITK antigen affinity purified polyclonal antibody (Catalog # A01385-3) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for ITK at approximately 72 kDa. The expected band size for ITK is at 72 kDa |
| Gel % | 10–12% |
| Load | 30 ug |
| Transfer | nitrocellulose membrane, 150 mA, 50–90 min |
| Membrane | nitrocellulose |
| Blocking | 5% non-fat milk / TBS, 1.5 h RT |
| Primary antibody | 0.5 µg/mL |
| Primary incubation | overnight at 4 °C |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 |
| Wash | TBS-0.1% Tween, 3 × 5 min |
| Detection | ECL |
| Exposure / imaging | Tanon 5200 |
| Observed band | 72 kDa |
Section 2
What Is the Expected ITK Western Blot Band Size?
ITK has a 71.8 kDa predicted mass and runs at the expected ~72 kDa, since it lacks glycosylation, cleavage, or disulfide-linked dimerization.
| single sharp band at approximately 72 kDa | matches full-length ITK's predicted mass of 71.8 kDa with no major post-translational mass addition |
| band running slightly higher than 72 kDa | phosphorylation at Tyr180, Tyr512, or Ser565 can retard migration slightly relative to the unmodified protein |
| faint higher-molecular-weight band above the main 72 kDa band | residual ITK homooligomers not fully dissociated by the sample buffer, since ITK self-associates and this is not disulfide-linked |
| band present in Jurkat/MOLT-4 lysates but absent in non-lymphoid lysates | ITK expression is restricted to T and NK lineage cells as part of TCR signaling |
| additional lower-molecular-weight bands beneath the main band | proteolytic degradation fragments of the kinase generated during lysis |
💡Expected ITK appearanceITK typically appears as a single sharp band at approximately 72 kDa, matching its 71.8 kDa predicted mass with no glycosylation- or cleavage-driven shift.
| Predicted mass (71.8 kDa) | sets the baseline monomer size, closely matching the observed ~72 kDa band |
| Phosphorylation at Tyr180, Tyr512, and Ser565 | can produce a slight upward mobility shift compared with the dephosphorylated form |
| Homooligomerization | may leave a faint higher-molecular-weight species if oligomers are not fully disrupted by reducing/denaturing sample buffer |
| No signal peptide or propeptide | ITK is not proteolytically processed, so the full-length protein runs at its predicted size with no smaller mature form |
| Lineage-restricted cytoplasmic/nuclear expression | band intensity depends heavily on cell type, being strong in T/NK cells and minimal or absent elsewhere |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | ITK expression is restricted to T and NK cells, so non-lymphoid lysates lack the protein | use a T-cell line such as Jurkat or MOLT-4 as a positive control lysate |
| Band higher than expected | incomplete dissociation of ITK homooligomers or a phosphorylation-associated mobility shift | boil samples longer in fresh reducing sample buffer and compare against a phosphatase-treated control |
| Multiple bands | partial degradation products or residual oligomeric species migrating alongside the ~72 kDa monomer | add fresh protease inhibitors during lysis and confirm the ~72 kDa band is the dominant species |
| Weak or no signal | low ITK abundance in cells with minimal TCR signaling activity or epitope masking by phosphorylation | stimulate T cells before lysis and increase total protein loaded per lane |
| Fragments below expected size | proteolytic cleavage of the kinase during sample preparation | keep lysates cold, add fresh protease inhibitors, and minimize freeze-thaw cycles |
Experiment evidence
Western blot images showing ITK band
A01385-3 · ITK
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for ITK Western blot
🧪For positive controls for ITK in Western blot, you can use HEK293 cells transiently transfected with an ITK expression construct, since no Human Protein Atlas tissue or cell-line expression data are available to identify an endogenous positive source for this protein.
Positive control: ITK-transfected HEK293 cells (overexpression)
Negative control: ubiquitously expressed; use siRNA knockdown or KO line
Loading controls: Run GAPDH and β-actin as loading controls alongside a total-protein stain such as stain-free imaging, Ponceau S, or REVERT.
⚠️Feasibility: No Human Protein Atlas expression data exist for ITK, so a validated positive or negative tissue cannot be confidently chosen and controls should instead rely on an overexpression system paired with siRNA/CRISPR knockdown or knockout confirmation.
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.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|
Section 3
Advanced ITK Western Blot Tips
Deeper troubleshooting and optimisation questions for ITK, answered from its protein features.
Why does ITK run close to its predicted 71.8 kDa?
ITK has no signal peptide, propeptide, glycosylation, or disulfide bonds, and only one annotated isoform, so nothing trims or modifies its backbone mass substantially. This explains why the observed band (~72 kDa) closely matches the predicted 71.8 kDa, with only the 3 modified residues contributing minor mass shifts.
Should multiple ITK isoforms appear on the blot?
No. UniProt lists only a single ITK isoform, so a clean lysate should show one dominant band near 72 kDa. Additional bands are more likely degradation products, incomplete denaturation of homooligomers, or nonspecific antibody binding rather than genuine isoform variation.
Does TCR stimulation change ITK phosphorylation detection?
ITK carries 3 modified residues consistent with activation-loop phosphorylation downstream of TCR engagement. Stimulating T cells (e.g., anti-CD3/CD28) before lysis increases phospho-ITK signal if using phospho-specific antibodies; unstimulated lysates may show weaker phospho-signal despite similar total ITK levels.
Which blocking buffer suits phospho-ITK detection?
Use BSA rather than milk-based blockers when probing ITK's phosphorylated residues. Milk contains casein phosphoproteins that cross-react with phospho-specific antibodies, elevating background and obscuring the true phospho-ITK band near 72 kDa.
What transfer method to use for ITK Western blot?
For a ~72 kDa cytoplasmic/nuclear kinase like ITK, standard wet (tank) transfer to PVDF with methanol-containing buffer gives reliable, efficient transfer. Semi-dry transfer also works well at this size; extended transfer times are unnecessary since ITK is not a large or membrane-embedded protein.
How to quantitate ITK across cytoplasm and nucleus?
Since ITK localizes to both cytoplasm and nucleus (redistributing to the plasma membrane upon receptor stimulation), subcellular fractionation with compartment-specific loading controls (e.g., GAPDH for cytoplasm, Lamin B1 for nucleus) gives more accurate quantitation than whole-cell lysate alone.
What explains higher molecular weight ITK bands?
ITK homooligomerizes, which negatively regulates its kinase activity; incomplete denaturation or reduction can leave residual oligomers migrating above 72 kDa. ITK is also subject to Ubl (ubiquitin-like) conjugation, which can produce a laddering pattern of higher-molecular-weight species distinct from the monomeric band.
Boster reagents
Best ITK Western Blot Antibodies
BosterBio's ITK antibodies are among the best-performing WB antibodies on the market — well cited, thoroughly validated, and orthogonally cross-validated against negative tissues and complementary methods.
Real WB data
Anti-ITK Antibody Picoband®
Cat # A01385-3
Our recommended anti-ITK antibodies are best-performing, well-cited reagents thoroughly validated for western blot, with specificity confirmed through orthogonal cross-validation against negative tissue and complementary detection methods—giving you confidence in reproducible, accurate ITK detection for your experiments.
Which to pick: Only one ITK antibody is catalogued here, A01385-3. It includes an actual western blot validation image showing specific ITK detection, making it the clear and only choice available for your ITK western blot work.
Source: BosterBio ITK gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q08881.
- Human Protein Atlas. ITK tissue expression.