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- Table of Contents
SF3B1 / Splicing factor 3B subunit 1 · Western blot design guide
Design a Western Blot for SF3B1
Real validated SF3B1 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-SF3B1 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
SF3B1 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 | ~145.8 kDa | |
| Observed band | ~155 kDa | |
| Gel | 8% (catalog M00507-1) | |
| Positive control | Bone marrow (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 + Acetylated | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated SF3B1 Western Blot Protocols
The M00507-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human HepG2, human Hela, human RT4 (catalog M00507-1) |
| Gel % | 8% (catalog M00507-1) |
| Load | 30 ug; reducing conditions (catalog M00507-1) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog M00507-1) |
| Membrane | nitrocellulose membrane (catalog M00507-1) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog M00507-1) |
| Primary antibody | M00507-1 · 1:500 (catalog M00507-1) |
| Primary incubation | overnight at 4°C (catalog M00507-1) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog M00507-1) |
| Secondary incubation | 1.5 hour at RT (catalog M00507-1) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog M00507-1) |
| Detection | ECL (catalog M00507-1) |
Section 2
What Is the Expected SF3B1 Western Blot Band Size?
SF3B1 is predicted at 145.8 kDa and observed at ~155 kDa; the cause of the difference is not established.
| Band at ~155 kDa | Empirical SF3B1 band in whole-cell lysate; confirm identity with antibody controls |
| Band near 145.8 kDa | Near the predicted SF3B1 mass; identity requires confirmation |
| Additional bands near the main band | Isoforms 1 and 2 are possible contributors, but distinct migration is unproven |
| Little or no band in a cytoplasmic fraction | SF3B1 is primarily nuclear and occurs in nuclear speckles |
💡Expected SF3B1 appearanceSF3B1 has a predicted mass of 145.8 kDa and an empirical band at ~155 kDa in whole-cell lysate; the difference is unexplained, so confirm band identity with appropriate antibody controls.
| Predicted SF3B1 mass | 145.8 kDa provides a sequence-based reference, while the empirical band is ~155 kDa |
| Isoforms 1 and 2 | May differ in size, but their individual masses and migration are unspecified |
| Phosphothreonine at residue 125 | A migration effect has not been established |
| Phosphoserine at residue 129 | A migration effect has not been established |
| Situation | Likely cause | Next action |
|---|---|---|
| Band higher than expected | The ~155 kDa empirical band exceeds the 145.8 kDa predicted mass for an undetermined reason | Compare with the validated whole-cell blot and confirm identity using an independent antibody or SF3B1 depletion |
| Band lower than expected | Isoform identity or sample fragmentation is uncertain | Check sample integrity and confirm the band using SF3B1 depletion or an independent antibody |
| Multiple bands | Isoforms 1 and 2 are annotated, but distinct bands are not established | Compare antibody epitopes and test band identity with SF3B1 depletion |
| Weak or no signal | Nuclear SF3B1 may be poorly recovered in the prepared sample | Check nuclear extraction and loading with a nuclear protein control |
| Fragments below expected size | Sample degradation is possible; fragment sizes are not specified | Improve sample handling and check whether smaller bands track with SF3B1 depletion |
Experiment evidence
Western blot images showing SF3B1 band
M00507-1 · SF3B1
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for SF3B1 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for SF3B1 in Western blot, you can use bone marrow tissue, which has high HPA expression.
Positive control: Bone marrow (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: SF3B1 is nuclear, so a nuclear-enriched lysate may improve its detection.
HPA tissue expression evidence for SF3B1
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 |
|---|---|---|---|---|
| Bone marrow | hematopoietic cells | High | Protein (IHC) | HPA → |
| Cerebral cortex | neuronal cells | High | Protein (IHC) | HPA → |
| Cervix | glandular cells | High | Protein (IHC) | HPA → |
| Gallbladder | glandular cells | High | Protein (IHC) | HPA → |
| Hippocampus | neuronal 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 → |
| Breast | adipocytes | Low | Protein (IHC) | HPA → |
| Thyroid gland | glandular cells | Low | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | Medium | Protein (IHC) | HPA → |
| Appendix | lymphoid tissue | Medium | Protein (IHC) | HPA → |
Section 3
Advanced SF3B1 Western Blot Tips
Deeper troubleshooting and optimisation questions for SF3B1, answered from its protein features.
How should SF3B1 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 an SF3B1 isoform produce a smaller band?
Isoforms · UniProt lists two isoforms. Isoform 2 replaces residues 140–144 and lacks residues 145–1304 of the canonical sequence, so a much smaller product is plausible. An unexpected small band cannot be assigned to isoform 2 from size alone; check whether the antibody recognizes its retained sequence.
Which SF3B1 phosphorylation sites have named enzymes?
PTM · In UniProt canonical numbering, CDK11 is listed for phosphothreonine at positions 211, 235, 313, and 328; DYRK1A is listed for phosphothreonine at 434. If comparing phosphosite antibodies or reports, check their numbering convention before matching sites. These annotations do not establish a detectable band shift.
UniProt lists acetyllysine at canonical positions 141, 214 (alternate), 554, and 562, plus citrulline at 157. Isoform 2 replaces the segment containing canonical Lys141 and lacks the later sites. These features suggest possible molecular differences, but they do not identify the cause of any particular band.
UniProt lists acetyllysine at canonical positions 141, 214 (alternate), 554, and 562, plus citrulline at 157. Isoform 2 replaces the segment containing canonical Lys141 and lacks the later sites. These features suggest possible molecular differences, but they do not identify the cause of any particular band.
Does this guide establish induction of SF3B1?
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 SF3B1 Western blot?
Transfer · The canonical protein is predicted at 145.8 kDa, with a reported apparent band near 155 kDa. Optimize transfer for that size and check whether protein remains in the gel after transfer. The supplied features do not establish one specific membrane or transfer setting.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the M00507-1 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 SF3B1 be quantified across sample preparations?
Quantitation · SF3B1 is annotated in the nucleus and nuclear speckles, with transient dispersal to the cytoplasm during mitosis. Compare like preparations and cell states, and use a loading reference appropriate to the fraction measured. A change in a nuclear fraction alone may reflect redistribution rather than a change in total SF3B1.
Why does SF3B1 appear near 155 kDa instead of 145.8 kDa?
Interpretation · The canonical sequence has a predicted mass of 145.8 kDa, while the reported apparent band is about 155 kDa. SF3B1 has many annotated modifications, but their presence alone does not explain the difference or establish a visible shift. Use the observed band as a reference and verify its identity if the distinction matters.
Consider the reported ~155-kDa canonical band, the much shorter isoform 2, and the annotated modifications when evaluating extra bands. None alone identifies an unexpected band or proves a shift. Check band identity with an antibody recognizing a different retained region or another protein-specific assay.
Consider the reported ~155-kDa canonical band, the much shorter isoform 2, and the annotated modifications when evaluating extra bands. None alone identifies an unexpected band or proves a shift. Check band identity with an antibody recognizing a different retained region or another protein-specific assay.
Boster reagents
SF3B1 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-SF3B1 Rabbit Monoclonal Antibody
Cat # M00507-1
The listed anti-SF3B1 antibody, M00507-1, has a Western blot image using human cell, rat brain and cell, and mouse brain and cell lysates. Its caption reports a band near 155 kDa versus an expected 146 kDa; evidence is limited to the supplied product image.
Which to pick: M00507-1 is the only listed option. It reports human, mouse, and rat reactivity and has a WB image with examples from each species. The caption used a 1:500 primary dilution; choose based on your sample and confirm the observed band in your own assay.
Source: BosterBio SF3B1 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry O75533.
- Human Protein Atlas. SF3B1 tissue expression.
- PMC4743009 — target-verified WB comparison
- PMC7139785 — target-verified WB comparison
- PMC11791315 — target-verified WB comparison