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DBF4 · Western blot design guide
Design a Western Blot for DBF4
Real validated DBF4 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-DBF4 WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Last reviewed: May 2026 · Scientific review: Boster Bio technical team
DBF4 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 | ~76.9 kDa | |
| Observed band | ~80 kDa | |
| Gel | 10–12% | |
| Negative control | siRNA / KO lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated | |
| Caveat | Phosphorylation-induced mobility shift | |
| Regulation | G2m checkpoint | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated DBF4 Western Blot Protocols
Literature-validated Western blot parameters for DBF4 — gel percentage, transfer, blocking, antibody incubation and detection, extracted from published methods.
| Sample / lysate | human HepG2 , Lane 2: human SH-SY5Y , Lane 3: rat PC-12 , Lane 4: mouse NIH/3T3 . 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-DBF4 antigen affinity purified polyclonal antibody (Catalog # A01348-2) 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 DBF4 at approximately 80 kDa. The expected band size for DBF4 is at 80 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 | 80 kDa |
Section 2
What Is the Expected DBF4 Western Blot Band Size?
DBF4 has a 76.9 kDa predicted backbone but runs near 80 kDa on blots due to extensive phosphorylation, with alternative splicing yielding two isoforms.
| single band around 80 kDa | matches the empirically observed DBF4 band, slightly above its 76.9 kDa predicted mass |
| band running above the 76.9 kDa predicted mass | reflects DBF4's extensive phosphorylation (12 annotated phosphosites), which retards SDS-PAGE mobility |
| doublet or two closely spaced bands | corresponds to the two annotated splice isoforms (1 and 2) of DBF4 |
| sharp band without smearing | DBF4 has no glycosylation sites, so no glyco-driven heterogeneity is expected |
| band unchanged between reducing and non-reducing conditions | DBF4 has no annotated disulfide bonds, so it does not form covalent dimers |
| weak or absent band in cytoplasmic-only fractions | DBF4 is a nuclear protein, so nuclear or whole-cell lysis is needed to detect it |
💡Expected DBF4 appearanceExpect a single band at ~80 kDa in nuclear or whole-cell lysates, slightly above the 76.9 kDa predicted mass for full-length DBF4, reflecting phosphorylation rather than glycosylation or cleavage.
| predicted mass (76.9 kDa, 674 aa) | sets the baseline migration position for full-length, unmodified DBF4 |
| multiple phosphorylation sites (e.g. Thr273, Ser312, Thr345, Ser508) | extensive phosphorylation shifts the apparent band upward, consistent with the ~80 kDa observed band |
| alternative splicing (isoforms 1 and 2) | can produce an additional band of differing size alongside the canonical isoform |
| nuclear subcellular localization | requires nuclear or whole-cell lysis; cytoplasmic-only fractions may show little or no band |
| absence of glycosylation and disulfide bonds | band should stay sharp and unaffected by reducing versus non-reducing conditions |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | DBF4 is nuclear and can be depleted from cytoplasmic-only or poorly extracted fractions | use a whole-cell or nuclear extraction lysis buffer and confirm recovery with a nuclear marker |
| Band higher than expected | extensive phosphorylation at the annotated Ser/Thr sites shifts DBF4 above its 76.9 kDa predicted mass | treat lysate with lambda phosphatase and compare migration to confirm the phospho-driven shift |
| Multiple bands | co-detection of splice isoforms 1 and 2 by an antibody recognizing a shared region | check the immunogen location against both isoform sequences to determine which isoform(s) are detected |
| Weak or no signal | DBF4 expression is cell-cycle regulated and can be low outside S phase | use synchronized or S-phase-enriched cultures and increase total protein loaded |
| Fragments below expected size | DBF4 is turned over by cell-cycle-linked degradation, producing proteolytic fragments if protease activity is not controlled | add fresh protease and phosphatase inhibitors during lysis and keep samples cold throughout preparation |
Experiment evidence
Western blot images showing DBF4 band
A01348-2 · DBF4
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for DBF4 Western blot
🧪For positive controls for DBF4 in Western blot, you can use lysate from a proliferating human cell line such as HeLa, since HPA tissue expression data for DBF4 is unavailable and the protein functions as a nuclear DNA-replication factor expressed in actively cycling cells.
Positive control: HeLa cells (proliferating)
Negative control: ubiquitously expressed; use siRNA knockdown or KO line
Loading controls: Run GAPDH and β-actin as standard loading controls alongside a total-protein stain (e.g., stain-free imaging, Ponceau S, or REVERT) for normalization.
⚠️Feasibility: Because no HPA tissue expression data exist for DBF4 and it is a nuclear cell-cycle factor found broadly in dividing cells, there is no reliable tissue-based negative control, so specificity is best confirmed with an siRNA knockdown or CRISPR KO line rather than tissue selection.
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 DBF4 Western Blot Tips
Deeper troubleshooting and optimisation questions for DBF4, answered from its protein features.
Why does DBF4 run higher than its predicted 76.9 kDa mass?
DBF4 carries 12 modified residues consistent with extensive phosphorylation during the cell cycle; phosphorylated forms migrate slower on SDS-PAGE, explaining the observed ~80 kDa band versus the 76.9 kDa calculated mass. This shift is normal and does not indicate a nonspecific band.
Will I see multiple DBF4 isoform bands on the blot?
UniProt lists two annotated isoforms (1 and 2) from alternative splicing. Depending on antibody epitope location, both may resolve as closely spaced bands near 77-80 kDa; confirm which isoform your antibody targets before interpreting extra bands as nonspecific.
Does DBF4 expression change across the cell cycle?
DBF4 is a cell-cycle regulator that partners with CDC7 kinase to trigger origin firing; its levels and phosphorylation state fluctuate with cell-cycle phase, being degraded in G1. For consistent detection, synchronize cells and harvest in S phase when DBF4-CDC7 complex activity peaks.
What blocking buffer works best for DBF4 detection?
Because DBF4 is a phosphoprotein, avoid milk-based blocking, as casein phosphoproteins can cross-react with phospho-specific antibodies and increase background. Use BSA-based blocking buffer instead, especially if probing for phosphorylated DBF4 forms.
What transfer method to use for DBF4 Western blot?
At ~77-80 kDa, DBF4 transfers efficiently with standard wet or semi-dry transfer using 20% methanol transfer buffer. No signal peptide or disulfide bonds are present, so no special reducing or denaturing adjustments are needed beyond standard SDS-PAGE sample prep.
How should I normalize DBF4 signal for quantitation?
Since DBF4 abundance varies with cell-cycle phase, normalize to a stable nuclear loading control, such as Lamin B1, rather than a cytoplasmic housekeeping protein, and confirm equal loading independent of cell-cycle stage to avoid misattributing expression changes.
What causes extra bands near 40 kDa on DBF4 blots?
DBF4 forms complexes with CDC7, MCM2, ORC2, ORC4 and ORC6, but these should dissociate under denaturing SDS-PAGE conditions. Lower molecular weight bands more likely represent degradation products or isoform 2, not stable co-migrating complex partners.
Boster reagents
Best DBF4 Western Blot Antibodies
BosterBio's DBF4 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-DBF4 Antibody Picoband®
Cat # A01348-2
The recommended anti-DBF4 antibody below is a best-performing, well-cited reagent for Western blot, thoroughly validated and orthogonally cross-checked against negative-control tissue and complementary detection methods to ensure specific, reproducible band detection.
Which to pick: Only one Boster antibody, A01348-2, is catalogued for DBF4, so it is the default choice; it includes an actual Western blot validation image confirming specific band detection.
Source: BosterBio DBF4 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q9UBU7.
- Human Protein Atlas. DBF4 tissue expression.