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- Table of Contents
TUBB4B / Tubulin beta-4B chain · Western blot design guide
Design a Western Blot for TUBB4B
Source-linked TUBB4B Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-TUBB4B 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
TUBB4B 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 | ~49.8 kDa | |
| Observed band | ~50 kDa | |
| Gel | 5–20% (catalog M09008) | |
| Positive control | Epididymis (IHC candidate; verify WB) +4 more | |
| Negative control | Cerebellum (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated + Acetylated | |
| Caveat | Paralog cross-reactivity | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 1 isoform(s) |
Section 1
Source-Linked TUBB4B Western Blot Protocol Options
The M09008 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human SH-SY5Y, rat brain, rat C6, mouse brain, mouse Neuro-2a (catalog M09008) |
| Gel % | 5–20% (catalog M09008) |
| Load | 30 ug; reducing conditions (catalog M09008) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog M09008) |
| Membrane | nitrocellulose membrane (catalog M09008) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog M09008) |
| Primary antibody | M09008 · 1:1000 (catalog M09008) |
| Primary incubation | overnight at 4°C (catalog M09008) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:500 (catalog M09008) |
| Secondary incubation | 1.5 hour at RT (catalog M09008) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog M09008) |
| Detection | ECL (catalog M09008) |
Section 2
What Is the Expected TUBB4B Western Blot Band Size?
TUBB4B is predicted at 49.8 kDa and observed at ~50 kDa; the supplied evidence does not establish a cause for the small difference.
| Dominant band at ~50 kDa | Matches the empirical band and the 49.8 kDa predicted mass. |
| Band enriched in cytoskeletal material | Consistent with TUBB4B localization to the cytoskeleton. |
| Faint band in a soluble fraction | May reflect partitioning of cytoskeletal TUBB4B during extraction. |
| No separate shifted band | The listed modification sites do not establish a resolvable mobility change. |
💡Expected TUBB4B appearanceTUBB4B has a predicted mass of 49.8 kDa and an empirical band at ~50 kDa; confirm band identity with appropriate antibody and sample controls.
| Predicted molecular mass | Places the unmodified chain near 49.8 kDa, consistent with the ~50 kDa empirical band. |
| Phosphothreonine at Thr55 | A visible mobility change is not established. |
| N6-acetyllysine at Lys58 | A visible mobility change is not established. |
| Phosphoserine at Ser172 | A visible mobility change is not established. |
| 5-glutamyl polyglutamate at residue 438 | Its effect on apparent band size is not established. |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Cytoskeletal TUBB4B may be poorly recovered by the extraction method. | Check the insoluble fraction and use a positive lysate control. |
| Band higher than expected | The listed modifications do not establish the cause of a higher band. | Compare reducing conditions and verify identity with an independent antibody. |
| Band lower than expected | A lower band is not explained by a listed signal peptide or propeptide. | Check sample integrity and verify identity with an independent antibody. |
| Multiple bands | The supplied UniProt record lists one isoform and does not explain extra bands. | Compare a positive control and confirm specificity with an independent antibody. |
| Weak or no signal | Extraction may leave cytoskeletal TUBB4B in the insoluble fraction. | Check fraction recovery and include a positive lysate control. |
| Fragments below expected size | Sample proteolysis is possible; no defined cleavage product is supplied. | Prepare fresh lysate with protease inhibitors and compare with a positive control. |
Experiment evidence
Western blot images showing TUBB4B band
M09008 · TUBB4B
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for TUBB4B Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for TUBB4B in Western blot, you can use epididymis tissue, which HPA scores as high expression.
Positive control: Epididymis (IHC candidate; verify WB)
Negative control: Cerebellum (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside the samples.
⚠️Feasibility: Cerebellum is reported as not detected by HPA, but confirm its negative signal in your lysate.
HPA tissue expression evidence for TUBB4B
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 |
|---|---|---|---|---|
| Epididymis | glandular cells | High | Protein (IHC) | HPA → |
| Fallopian tube | ciliated cells (cilia axoneme) | High | Protein (IHC) | HPA → |
| Lymph node | germinal center cells | High | Protein (IHC) | HPA → |
| Nasopharynx | ciliated cells (cilia axoneme) | High | Protein (IHC) | HPA → |
| Skin | cells in granular layer | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Cerebellum | cells in granular layer | Not detected | Protein (IHC) | HPA → |
| Heart muscle | cardiomyocytes | Not detected | Protein (IHC) | HPA → |
| Parathyroid gland | glandular cells | Not detected | Protein (IHC) | HPA → |
| Placenta | decidual cells | Not detected | Protein (IHC) | HPA → |
| Skeletal muscle | myocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced TUBB4B Western Blot Tips
Deeper troubleshooting and optimisation questions for TUBB4B, answered from its protein features.
Where should the main TUBB4B band appear?
Band shift · TUBB4B has a predicted mass of 49.8 kDa, and the supplied observed band is approximately 50 kDa. Use that region as the starting point for band identification.
Could an annotated isoform account for another band?
Isoforms · The supplied record lists one isoform and no alternative sequence. It therefore provides no annotated isoform to assign to an additional band.
Do TUBB4B modifications explain a shifted band?
PTM · UniProt lists phosphothreonine at residue 55, N6-acetyllysine at 58, CDK1-dependent phosphoserine at 172, and 5-glutamyl polyglutamate at 438. These are UniProt coordinates; antibody or paper numbering may differ. Their presence alone does not establish a visible shift or explain a mass discrepancy.
UniProt identifies CDK1 for phosphorylation of serine 172. This is the supplied UniProt coordinate; check numbering conventions before comparing it with an antibody or paper. The record does not establish when the site is occupied in your sample.
UniProt identifies CDK1 for phosphorylation of serine 172. This is the supplied UniProt coordinate; check numbering conventions before comparing it with an antibody or paper. The record does not establish when the site is occupied in your sample.
Does this guide establish induction of TUBB4B?
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 TUBB4B?
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 M09008 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 I quantify the approximately 50 kDa band?
Quantitation · Measure the same approximately 50 kDa region consistently across samples. The listed phosphorylation, acetylation, and polyglutamylation sites do not establish modification occupancy from total band intensity.
What should I consider when examining TUBB4B's C-terminal region?
Interpretation · UniProt lists 5-glutamyl polyglutamate at residue 438, near the end of the 445-residue sequence. Keep that UniProt coordinate when comparing site-specific results; the listing alone does not predict a distinct Western-blot band.
Compare them with the approximately 50 kDa observed band. The supplied record lists one isoform, no signal peptide or propeptide, and no glycosylation sites. Its listed modifications do not, by themselves, identify the cause of an extra band.
Compare them with the approximately 50 kDa observed band. The supplied record lists one isoform, no signal peptide or propeptide, and no glycosylation sites. Its listed modifications do not, by themselves, identify the cause of an extra band.
Boster reagents
TUBB4B 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-Tubulin beta 4B/2A/2B Rabbit Monoclonal Antibody
Cat # M09008
M09008 is a rabbit monoclonal antibody for Tubulin beta 4B/2A/2B with a Western blot image showing an approximately 50 kDa band in the listed human, rat, and mouse samples. The supplied evidence does not establish TUBB4B-specific detection.
Which to pick: M09008 is the only listed option. Its WB image includes human SH-SY5Y cells, rat brain and C6 cells, and mouse brain and Neuro-2a cells. Choose it for those reported contexts, while accounting for its combined beta 4B/2A/2B target designation.
Source: BosterBio TUBB4B gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P68371.
- Human Protein Atlas. TUBB4B tissue expression.
- PMC6721557 — target-verified WB comparison
- PMC9222938 — target-verified WB comparison
- PMC12886888 — target-verified WB comparison