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CBFB · Western blot design guide
Design a Western Blot for CBFB
Real validated CBFB Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-CBFB WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Last reviewed: May 2026 · Scientific review: Boster Bio technical team
CBFB 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 | ~21.5 kDa | |
| Observed band | ~22 kDa | |
| Gel | 12–15% | |
| Negative control | siRNA / KO lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated | |
| Caveat | Alternative splicing isoforms | |
| Regulation | IFN-γ-induced | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated CBFB Western Blot Protocols
Literature-validated Western blot parameters for CBFB — gel percentage, transfer, blocking, antibody incubation and detection, extracted from published methods.
| Sample / lysate | human SW620 , Lane 2: rat thymus , Lane 3: mouse thymus . After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA 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-CBFb antigen affinity purified polyclonal antibody (Catalog # A01007-1) 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 1:10000 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 CBFb at approximately 22KD. The expected band size for CBFb is at 22KD |
| Gel % | 12–15% |
| Load | 50ug |
| 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, 1:10000 |
| Wash | TBS-0.1% Tween, 3 × 5 min |
| Detection | ECL |
| Exposure / imaging | Tanon 5200 |
| Observed band | 22 kDa |
Section 2
What Is the Expected CBFB Western Blot Band Size?
CBFB's 21.5 kDa predicted mass matches its ~22 kDa observed band, since it lacks glycosylation, disulfide bonds, and cleavage; isoform splicing is the main source of extra bands.
| Single sharp band at ~22 kDa | Matches CBFB's unmodified 21.5 kDa predicted mass, with no glycosylation or disulfide-linked shift |
| Faint second band a few kDa away from the main ~22 kDa band | Reflects co-expression of CBFB isoform 2 alongside canonical isoform 1 from alternative splicing |
| No mass change comparing reducing vs non-reducing conditions | CBFB has no annotated disulfide bonds, so it runs as a monomer regardless of reduction state |
| No higher-MW smear above the ~22 kDa band | CBFB has no annotated N- or O-glycosylation sites, so heterogeneous glycoform smearing is not expected |
| Detectable band directly in whole-cell or tissue lysates without concentration steps | CBFB is a nuclear protein, not secreted, so it is retained intracellularly and captured in standard lysates |
💡Expected CBFB appearanceExpect a single sharp band at approximately 22 kDa in whole-cell or nuclear lysates, matching CBFB's unmodified 21.5 kDa predicted mass, since there is no glycosylation, disulfide linkage, or signal-peptide cleavage to shift its size.
| Predicted mass (21.5 kDa, 182 aa) | Sets the baseline unmodified band position, closely matching the ~22 kDa band seen empirically |
| Absence of glycosylation | Rules out higher-MW smearing or upward shift from sugar addition, keeping the band sharp and near predicted size |
| Absence of disulfide bonds | No ~2x mass shift between reducing and non-reducing conditions; CBFB runs as a monomer, not a covalent dimer |
| Absence of signal peptide or propeptide | No precursor-to-mature cleavage, so there is no smaller processed band relative to the full-length protein |
| Alternative splicing (isoforms 1 and 2) | May produce a minor additional band of different apparent size alongside the dominant isoform 1 band |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | CBFB is a nuclear protein and standard cytoplasmic-lysis buffers can under-extract nuclear/chromatin-associated factors | Use a nuclear extraction buffer or add sonication/nuclease treatment to fully release CBFB before loading |
| Band higher than expected | Incomplete denaturation can leave CBFB associated with its RUNX1/2/3 heterodimer partner | Ensure samples are fully denatured with SDS sample buffer and heated before loading to dissociate the complex |
| Band lower than expected | Proteolytic degradation of the small 21.5 kDa CBFB protein during lysate preparation | Add protease inhibitors and keep samples cold throughout lysis and handling to limit degradation |
| Multiple bands | Co-detection of CBFB isoform 2 alongside the canonical isoform 1 from alternative splicing | Check which isoform the antibody epitope covers and compare relative isoform expression across sample types |
| Weak or no signal | Low endogenous CBFB expression relative to abundant housekeeping proteins in some tissue types | Load more total protein or include a known positive control such as thymus tissue lysate |
Experiment evidence
Western blot images showing CBFB band
A01007-1 · CBFB
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for CBFB Western blot
🧪For positive controls for CBFB in Western blot, you can use a nuclear-enriched lysate from a hematopoietic or proliferating cell line (e.g., HEK293 or Jurkat) known to express core-binding factor subunits, since Human Protein Atlas tissue/cell expression data are not available for this gene.
Positive control: HEK293 nuclear lysate
Negative control: ubiquitously expressed; use siRNA knockdown or KO line
Loading controls: Run GAPDH, β-actin, and a total-protein stain (e.g., stain-free gel, Ponceau S, or REVERT) alongside as loading controls.
⚠️Feasibility: No HPA expression data are available for CBFB, so tissue-based positive/negative selection is unreliable; confirm antibody specificity with siRNA knockdown or a CRISPR KO line and enrich for nuclear fractions given its nuclear localization.
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 CBFB Western Blot Tips
Deeper troubleshooting and optimisation questions for CBFB, answered from its protein features.
Why does CBFB run slightly heavier than its predicted mass?
Predicted mass is 21.5 kDa; observed band runs near 22 kDa, a normal small shift. CBFB is annotated as a phosphoprotein, and phosphorylation can slightly retard migration on SDS-PAGE even without a large mass change. No glycosylation or disulfide bonds are annotated, so the shift is not due to those modifications.
Does CBFB show two isoform-specific bands?
UniProt lists two CBFB isoforms generated by alternative splicing. Depending on antibody epitope location, both isoforms may be detected as closely spaced bands near 22 kDa, or only the canonical isoform if the epitope is spliced out. Confirm antibody immunogen sequence against both isoform sequences before interpreting multiple close bands as nonspecific.
Is CBFB expression tied to hematopoietic differentiation signals?
CBFB itself has no catalytic activity but functions as an obligate heterodimeric partner of RUNX1, RUNX2 and RUNX3, forming core-binding factor complexes essential for hematopoietic and skeletal gene regulation. Band intensity can vary with differentiation state of hematopoietic or osteogenic cell models, reflecting changes in RUNX-CBFB complex demand rather than direct induction of CBFB alone.
What blocking buffer avoids interference with phospho-CBFB detection?
Since CBFB is annotated as a phosphoprotein, use BSA-based blocking (5% BSA in TBST) rather than milk if probing with a phospho-specific antibody, as milk casein is itself phosphorylated and can cause high background or false signal with phospho-antibodies. Standard milk blocking is acceptable for total CBFB antibodies.
What transfer method to use for CBFB Western blot?
CBFB is a small (~21.5 kDa, observed ~22 kDa) nuclear protein, so use 0.2 micron PVDF or nitrocellulose with a shorter transfer time to prevent blow-through. Semi-dry or wet transfer at standard voltage both work well for this size range; monitor transfer efficiency with prestained markers near 20-25 kDa.
Which loading control best normalizes CBFB quantitation?
CBFB is nuclear-localized, part of the core-binding factor complex with RUNX proteins. For accurate quantitation, normalize to a nuclear loading control such as Lamin B or Histone H3 rather than a cytoplasmic marker like GAPDH, since nuclear extraction efficiency can vary independently of total cytoplasmic protein.
Why might a larger band appear in leukemia samples?
CBFB is subject to chromosomal rearrangement, most notably the CBFB-MYH11 fusion in inv(16) acute myeloid leukemia. In such samples, an antibody recognizing the CBFB N-terminus may detect an aberrant higher-molecular-weight fusion protein band in addition to, or instead of, the normal ~22 kDa wild-type band.
Boster reagents
Best CBFB Western Blot Antibodies
BosterBio's CBFB 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-CBFb Antibody Picoband®
Cat # A01007-1
Our recommended anti-CBFB antibody is a top-performing, extensively cited reagent, rigorously validated across applications and cross-checked against negative tissue controls and orthogonal methods for specificity, giving you confidence in every Western blot result.
Which to pick: Only one CBFB antibody is catalogued, A01007-1, backed by a real Western blot validation image showing the expected band, making it the clear, single choice for your CBFB Western blot experiments.
Source: BosterBio CBFB gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q13951.
- Human Protein Atlas. CBFB tissue expression.