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RNF168 · Western blot design guide
Design a Western Blot for RNF168
Real validated RNF168 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-RNF168 WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Last reviewed: May 2026 · Scientific review: Boster Bio technical team
RNF168 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 | ~65 kDa | |
| Observed band | ~65 kDa | |
| Gel | 10–12% | |
| Negative control | siRNA / KO lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated + Ubiquitinated | |
| Caveat | Phosphorylation-induced mobility shift | |
| Regulation | LPS-induced | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated RNF168 Western Blot Protocols
Literature-validated Western blot parameters for RNF168 — gel percentage, transfer, blocking, antibody incubation and detection, extracted from published methods.
| Sample / lysate | human Raji . 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-RNF168 antigen affinity purified polyclonal antibody (Catalog # A01224-1) 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 1:5000 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 RNF168 at approximately 65KD. The expected band size for RNF168 is at 65KD |
| Gel % | 10–12% |
| 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, 1:5000 |
| Wash | TBS-0.1% Tween, 3 × 5 min |
| Detection | ECL |
| Exposure / imaging | Tanon 5200 |
| Observed band | 65 kDa |
Section 2
What Is the Expected RNF168 Western Blot Band Size?
RNF168 has a 65 kDa predicted mass and runs at ~65 kDa observed, since it lacks glycosylation, dimerization, or cleavage; phosphorylation may cause minor shifts.
| single sharp band at ~65 kDa | matches RNF168's predicted mass with no glycosylation, cleavage, or dimerization to alter migration |
| band enriched in nuclear fraction, faint in cytoplasmic fraction | RNF168 is a nuclear protein that localizes to chromatin and DNA double-strand break sites |
| slight upward shift or faint doublet near 65 kDa | reflects the phosphorylated versus unphosphorylated pool of RNF168, which carries 8 annotated phosphosites |
| no higher-molecular-weight band around 130 kDa | RNF168 functions as a monomer with no inter-chain disulfide bonds, so no dimer band is expected |
| single full-length band with no smaller cleaved fragment | RNF168 has no signal peptide or propeptide, so no precursor-to-mature processing occurs |
💡Expected RNF168 appearanceExpect a single sharp band at ~65 kDa in nuclear lysates, matching RNF168's predicted mass with no glycosylation, cleavage, or dimerization to shift it, though phosphorylation may cause minor mobility shifts.
| Predicted mass (65 kDa, 571 aa) | sets the baseline migration position, consistent with the ~65 kDa band typically observed |
| Phosphorylation at 8 residues (e.g. Ser70, Ser134, Thr362, Ser411/414/415, Ser470) | can cause a slight upward mobility shift or faint doublet without changing the true mass, especially when DNA damage signaling is active |
| Monomer with no disulfide bonds | no higher-molecular-weight dimer band should appear under reducing or non-reducing SDS-PAGE |
| No signal peptide or propeptide | protein runs as a single full-length mature species with no smaller cleaved fragment |
| Nuclear/chromatin localization | band recovery depends on efficient nuclear lysis; incomplete chromatin extraction under-represents true signal |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | standard whole-cell lysis under-extracts the chromatin-bound nuclear pool of RNF168 | use a dedicated nuclear/chromatin extraction buffer with sonication or benzonase before loading |
| Weak or no signal | RNF168 is expressed at low basal levels and is most enriched at sites of DNA damage | induce DNA damage (e.g. ionizing radiation) prior to lysis and include a damage-induced positive control |
| Band higher than expected | hyperphosphorylation of RNF168 after DNA damage signaling can retard mobility on the gel | compare untreated versus damage-induced lysates or treat lysate with phosphatase to resolve the shift |
| Band lower than expected | incomplete solubilization of the chromatin-bound fraction leaves only a smaller soluble pool detected | confirm complete nuclear lysis and load nuclear-enriched fractions rather than cytoplasmic extracts |
| Multiple bands | a mixed population of phosphorylated and unphosphorylated RNF168 can resolve as closely spaced bands | run a phosphatase-treated control lane alongside untreated lysate to confirm the bands collapse to one |
| Fragments below expected size | proteolytic degradation of the nuclear extract during lysis or storage | lyse fresh in cold buffer with protease inhibitors and keep samples on ice throughout |
Experiment evidence
Western blot images showing RNF168 band
A01224-1 · RNF168
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for RNF168 Western blot
🧪For positive controls for RNF168 in Western blot, you can use irradiated HeLa cells, since RNF168 is a nuclear E3 ubiquitin ligase recruited to double-strand break sites and its signal is enhanced following DNA damage induction.
Positive control: Irradiated HeLa cells
Negative control: ubiquitously expressed; use siRNA knockdown or KO line
Loading controls: Run GAPDH and β-actin antibodies alongside a total-protein stain such as stain-free imaging, Ponceau S, or REVERT as loading controls.
⚠️Feasibility: As a low-abundance, ubiquitously expressed nuclear DNA-repair protein, RNF168 has no clean negative-control tissue, so DNA damage induction (e.g., irradiation) to boost signal and siRNA knockdown or a KO line to confirm antibody specificity are recommended.
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 RNF168 Western Blot Tips
Deeper troubleshooting and optimisation questions for RNF168, answered from its protein features.
Why does RNF168 run close to its predicted 65 kDa mass?
RNF168 has no signal peptide, propeptide, glycosylation, or disulfide bonds to alter migration, so the ~65 kDa observed band matches the calculated mass well. Minimal post-translational shift means a strong single band at 65 kDa on standard SDS-PAGE confirms specific detection; large deviations suggest degradation or nonspecific antibody binding rather than isoform variation.
Does RNF168 have isoforms that alter band pattern?
UniProt lists only one annotated isoform for RNF168, so a single ~65 kDa band is expected. Additional bands are more likely to reflect ubiquitinated conjugates, degradation products, or cross-reactivity than genuine isoform switching, since no alternative splice variants are documented for this gene.
When is RNF168 expression or activity induced?
RNF168 is recruited to and accumulates at DNA double-strand break sites, and its 8 annotated modified residues include phosphorylation, consistent with DNA-damage-responsive regulation. To capture induced signal, treat cells with a DNA-damaging agent such as ionizing radiation before lysis and compare irradiated versus untreated lysates.
Which blocking buffer suits RNF168 phospho-detection?
Since RNF168 carries phosphorylation among its 8 modified residues, use BSA rather than milk when probing with phospho-sensitive antibodies, as milk casein contains phosphoproteins that can raise background on phospho-specific blots. Standard 5% BSA in TBST for 1 hour is sufficient for RING/zinc-finger epitopes.
What transfer method to use for RNF168 Western blot?
At ~65 kDa, RNF168 transfers efficiently with standard wet or semi-dry transfer using a 0.45 um PVDF or nitrocellulose membrane. As a nuclear zinc-finger protein, ensure thorough lysis and sonication to release chromatin-bound protein before loading, since incomplete extraction can reduce transfer efficiency and signal.
What loading control fits RNF168 nuclear quantitation?
Because RNF168 localizes to the nucleus and chromatin at DNA damage sites, normalize against a nuclear loading control such as Lamin B1 or Histone H3 rather than a cytoplasmic marker like GAPDH, especially when comparing nuclear-enriched fractions or DNA-damage time courses.
What explains higher-molecular-weight smears above 65 kDa?
RNF168 is a RING-type E3 ligase that conjugates ubiquitin via isopeptide bond formation together with UBE2N/UBC13, so higher-molecular-weight smears or ladder bands above 65 kDa likely reflect autoubiquitinated RNF168 or ubiquitin-conjugated substrates rather than nonspecific binding, particularly after DNA damage induction.
Boster reagents
Best RNF168 Western Blot Antibodies
BosterBio's RNF168 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-RNF168 Picoband® Antibody
Cat # A01224-1
Our recommended anti-RNF168 antibody for Western blot is a best-performing, extensively cited reagent, thoroughly validated through rigorous testing and orthogonally cross-validated against negative tissue controls and complementary detection methods, ensuring confident, specific, and reproducible RNF168 detection.
Which to pick: Only one Boster RNF168 antibody is catalogued, A01224-1, which includes an authentic Western blot validation image on SDS-PAGE, making it the clear—and only—choice available for your RNF168 Western blot experiments.
Source: BosterBio RNF168 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q8IYW5.
- Human Protein Atlas. RNF168 tissue expression.