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- Table of Contents
IBTK / Inhibitor of Bruton tyrosine kinase · Western blot design guide
Design a Western Blot for IBTK
Source-linked IBTK Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-IBTK WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Evidence assembled October 2026 · For research use; verify linked source records and product datasheet before use
IBTK 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 | ~150.5 kDa | |
| Gel | 8–10% (standard starting point) | |
| Positive control | Appendix (IHC candidate; verify WB) +4 more | |
| Negative control | Oral mucosa (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 | 3 isoform(s) |
Section 1
Source-Linked IBTK Western Blot Protocol Options
The A07985 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | extracts of various cell lines, (catalog A07985) |
| Gel % | 8–10% (standard starting point) |
| Load | 20–30 µg total protein per lane; optimize for abundance (standard starting point) |
| Transfer | Wet/tank transfer; optimize duration (standard starting point) |
| Membrane | 0.45 µm PVDF (standard starting point) |
| Blocking | Blocking buffer: 3% nonfat dry milk in TBST (catalog A07985) |
| Primary antibody | A07985 · 1:1000 (catalog A07985) |
| Primary incubation | Overnight at 4 °C (standard starting point) |
| Secondary antibody | Goat Anti-Rabbit IgG, 1:10000 (catalog A07985) |
| Secondary incubation | 1 h at room temperature (standard starting point) |
| Wash | 3 × 5 min in TBST (standard starting point) |
| Detection | ECL (catalog A07985) |
Section 2
What Is the Expected IBTK Western Blot Band Size?
IBTK is predicted at 150.5 kDa; isoforms and phosphorylation could affect migration, but no empirical band or demonstrated shift is supplied.
| Band near 150.5 kDa | Consistent with the predicted IBTK mass; confirm identity with antibody controls. |
| Multiple bands near the expected region | Could reflect isoforms 1, 2, and 3; their migration differences are unknown. |
| Slightly shifted band or doublet | Could reflect phosphorylation; a visible shift is not established. |
| Reduced cytoplasmic band after IgM stimulation | Consistent with IBTK translocation to the plasma membrane. |
💡Expected IBTK appearanceUniProt predicts 150.5 kDa for IBTK, but no empirical band size is supplied; assess bands near that size with antibody and sample controls because isoform migration and phosphorylation effects are unquantified.
| UniProt predicted mass | Places the reference protein near 150.5 kDa. |
| Isoform 1 | Its individual mass and migration are not supplied. |
| Isoform 2 | Alternative splicing may change migration, but its size is unspecified. |
| Isoform 3 | Alternative splicing may change migration, but its size is unspecified. |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | IBTK may redistribute to the plasma membrane after IgM stimulation, or detection may be insufficient. | Check membrane and cytoplasmic fractions and verify antibody detection with a positive control. |
| Band higher than expected | Phosphorylation could affect migration, but the size of any shift is unknown. | Compare phosphatase-treated material and confirm band identity with a second antibody. |
| Band lower than expected | An alternatively spliced isoform is possible; isoform masses are unavailable. | Confirm identity with an independent antibody or isoform-specific sample. |
| Multiple bands | IBTK has three named isoforms and documented phosphorylation sites; distinct bands are not established. | Compare isoform-specific samples or phosphatase-treated material and confirm antibody specificity. |
| Weak or no signal | IgM-dependent redistribution may reduce IBTK in a sampled fraction. | Check whole-cell and membrane fractions alongside a positive control. |
Experiment evidence
Western blot images showing IBTK band
A07985 · IBTK
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for IBTK Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for IBTK in Western blot, you can use appendix lysate, which has medium HPA staining.
Positive control: Appendix (IHC candidate; verify WB)
Negative control: Oral mucosa (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: IBTK is cytoplasmic and membrane-associated, so whole-tissue lysate is suitable; oral mucosa provides an HPA not-detected comparator.
HPA tissue expression evidence for IBTK
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 | Medium | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | Medium | Protein (IHC) | HPA → |
| Cerebellum | Purkinje cells | Medium | Protein (IHC) | HPA → |
| Cerebral cortex | neuronal cells | Medium | Protein (IHC) | HPA → |
| Cervix | glandular cells | Medium | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Oral mucosa | squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Smooth muscle | smooth muscle cells | Not detected | Protein (IHC) | HPA → |
| Soft tissue | chondrocytes | Not detected | Protein (IHC) | HPA → |
| Spleen | cells in red pulp | Not detected | Protein (IHC) | HPA → |
| Vagina | squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced IBTK Western Blot Tips
Deeper troubleshooting and optimisation questions for IBTK, answered from its protein features.
How should IBTK 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.
How might IBTK isoforms affect band interpretation?
Isoforms · UniProt lists three isoforms. Relative to the canonical sequence, isoform 2 replaces residues 1145–1196 and lacks 1197–1353; isoform 3 lacks 1–1113 and replaces 1114–1144. Check which sequence your antibody recognizes before assigning a band to an isoform.
Which IBTK phosphorylation sites matter when assessing bands?
PTM · UniProt lists phosphoserines at canonical positions 990, 1004, 1030, 1033, 1039, 1045, 1054, 1083, 1111, 1113, and 1116. These are UniProt coordinates and may differ from antibody or paper numbering. Their presence alone does not establish a visible band shift.
Does IgM stimulation change where IBTK is detected?
Induction · UniProt reports that IBTK translocates to the plasma membrane upon IgM stimulation. Compare matched cytoplasmic and membrane fractions when assessing that response; this feature does not establish an increase in total IBTK abundance.
Specify whether the measurement is total IBTK or its distribution between cytoplasm and membrane. UniProt reports plasma membrane translocation upon IgM stimulation, so compare matched fractions to assess redistribution.
Specify whether the measurement is total IBTK or its distribution between cytoplasm and membrane. UniProt reports plasma membrane translocation upon IgM stimulation, so compare matched fractions to assess redistribution.
What transfer method to use for IBTK Western blot?
Transfer · Canonical IBTK is predicted at 150.5 kDa. Choose and verify transfer conditions that retain detectable signal in that molecular weight range. The supplied features do not establish one specific transfer method.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A07985 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 IBTK 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 IBTK migrate at its predicted 150.5 kDa?
Interpretation · Use 150.5 kDa as the predicted reference for canonical IBTK. No observed band size is supplied. The listed phosphorylation sites and isoforms do not, by themselves, establish a visible shift or explain a difference from calculated mass.
Check antibody coverage of the three isoforms before assigning extra bands. Isoform 2 changes the sequence after position 1144 and lacks 1197–1353; isoform 3 lacks 1–1113 and replaces 1114–1144. Phosphorylation is annotated, but neither it nor an isoform annotation alone identifies an observed band.
Check antibody coverage of the three isoforms before assigning extra bands. Isoform 2 changes the sequence after position 1144 and lacks 1197–1353; isoform 3 lacks 1–1113 and replaces 1114–1144. Phosphorylation is annotated, but neither it nor an isoform annotation alone identifies an observed band.
Boster reagents
IBTK 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-IBTK Antibody
Cat # A07985
A07985 is a human-reactive anti-IBTK antibody with a Western blot image from cell-line extracts. The supplied record reports a 1:1000 primary dilution and 25 µg protein per lane; it provides no publication or independent validation evidence.
Which to pick: A07985 is the only listed IBTK antibody. It has a Western blot image and reported human reactivity; use the stated 1:1000 dilution as a starting point for your samples.
Source: BosterBio IBTK gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q9P2D0.
- Human Protein Atlas. IBTK tissue expression.
- PMC2490745 — target-verified WB comparison
- PMC6459904 — target-verified WB comparison
- PMC8875111 — target-verified WB comparison