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RAD51C · Western blot design guide
Design a Western Blot for RAD51C
Real validated RAD51C Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-RAD51C WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Last reviewed: May 2026 · Scientific review: Boster Bio technical team
RAD51C 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 | ~42.2 kDa | |
| Observed band | ~42 kDa | |
| Gel | 10–12% | |
| Negative control | siRNA / KO lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated | |
| Caveat | Alternative splicing isoforms | |
| Regulation | p53 up | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated RAD51C Western Blot Protocols
Literature-validated Western blot parameters for RAD51C — gel percentage, transfer, blocking, antibody incubation and detection, extracted from published methods.
| Sample / lysate | human Hela , Lane 2: human 293T , Lane 3: human A549 , Lane 4: rat testis . 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-RAD51C antigen affinity purified polyclonal antibody (Catalog # A01837-1) 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 RAD51C at approximately 42 kDa. The expected band size for RAD51C is at 42 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 | 42 kDa |
Section 2
What Is the Expected RAD51C Western Blot Band Size?
RAD51C has a 42.2 kDa predicted backbone and runs at the matching ~42 kDa observed band, since it lacks glycosylation and disulfide-linked oligomerization that would shift its size.
| single sharp band at ~42 kDa | matches the 42.2 kDa predicted mass, consistent with a protein carrying no glycosylation, disulfide bonds, or cleavable signal/propeptide sequence |
| band unchanged between reducing and non-reducing sample buffer | RAD51C has no inter-chain disulfide bonds, so there is no shift to a higher-mass covalent dimer under non-reducing conditions |
| band still runs at ~42 kDa monomer size even from lysates where RAD51C is complexed with other RAD51 paralogs | participation in the non-covalent BCDX2 and CX3 paralog complexes is disrupted by SDS and heat, so the protein resolves as a monomer rather than a high-molecular-weight complex band |
| faint second band close to the main ~42 kDa band | alternative splicing produces isoform 2 in addition to canonical isoform 1, which can appear as a closely spaced or minor additional band |
| slight upward smear or doublet around the main band | phosphorylation at Ser20 can create a small population of protein migrating marginally slower than the unmodified species |
| band present in both nuclear-enriched and whole-cell lysate fractions at the same size | RAD51C localizes to nucleus, cytoplasm, and mitochondrion without size-altering compartment-specific processing |
💡Expected RAD51C appearanceRAD51C typically appears as a single sharp band at approximately 42 kDa, closely matching its 42.2 kDa predicted mass, since the protein has no glycosylation, disulfide bonds, or cleavable signal or propeptide sequence.
| predicted mass (42.2 kDa) | sets the baseline expected migration, closely matched by the empirical ~42 kDa observed band |
| absence of glycosylation | no added carbohydrate mass, so the band stays near the predicted size rather than running higher or smearing |
| absence of disulfide bonds | protein migrates as a ~42 kDa monomer on both reducing and non-reducing gels, with no shift to an ~84 kDa dimer band |
| non-covalent BCDX2/CX3 complex membership | complex is dissociated by SDS and reducing/denaturing conditions, so the protein still runs as a single monomer band despite assembling into multi-subunit complexes in vivo |
| alternative splicing (isoforms 1 and 2) | canonical isoform 1 and shorter isoform 2 can each contribute a band, with isoform 2 expected to run somewhat differently without an exact assigned kDa |
| Ser20 phosphorylation | phosphorylated fraction can migrate marginally slower than the unmodified species, adding subtle heterogeneity around the main band |
| Situation | Likely cause | Next action |
|---|---|---|
| Band higher than expected | Ser20 phosphorylation or incomplete denaturation of paralog-complex-associated RAD51C | ensure complete sample denaturation with fresh reducing sample buffer and sufficient boiling, and use a phosphatase-treated control lane if a shift is suspected |
| Weak or no signal | RAD51C is a low-abundance nuclear DNA repair protein whose levels rise after DNA damage | treat cells with a genotoxic agent to induce expression, load more total protein, and use a nuclear-enriched extraction protocol |
| Multiple bands | detection of both splice isoforms 1 and 2, or degradation of the low-abundance protein | compare band pattern to isoform sequences and antibody epitope location, and add protease inhibitors to rule out degradation |
| Band lower than expected | proteolytic degradation of this low-abundance, DNA-damage-responsive protein during lysis | use fresh lysates with protease inhibitors and minimize sample handling and freeze-thaw cycles |
| No band in lysate | cytoplasm-only lysis protocols can under-recover RAD51C, which is concentrated in the nucleus and mitochondrion | use a whole-cell or nuclear/mitochondrial extraction protocol that efficiently solubilizes these compartments |
Experiment evidence
Western blot images showing RAD51C band
A01837-1 · RAD51C
PB9792 · RAD51C
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for RAD51C Western blot
🧪For positive controls for RAD51C in Western blot, you can use HeLa cell lysate, since RAD51C is a ubiquitously expressed DNA repair protein active in proliferating cells and no tissue-specific HPA expression data are available for this target.
Positive control: HeLa cells
Negative control: ubiquitously expressed; use siRNA knockdown or KO line
Loading controls: Run GAPDH and β-actin as loading controls alongside a total-protein stain such as stain-free gel, Ponceau S, or REVERT.
⚠️Feasibility: RAD51C is a low-abundance nuclear/cytoplasmic/mitochondrial DNA repair protein with no HPA tissue data to guide a clean negative tissue, so a genetic negative control (siRNA knockdown or CRISPR KO) is more reliable than tissue selection alone.
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 RAD51C Western Blot Tips
Deeper troubleshooting and optimisation questions for RAD51C, answered from its protein features.
Why might the RAD51C band differ from the 42 kDa prediction?
RAD51C runs close to its predicted 42.2 kDa mass, and the observed ~42 kDa band confirms minimal shift. Any deviation is more likely due to gel percentage or migration artifacts than major PTMs, since the protein carries only one modified residue and lacks glycosylation or disulfide bonds that would otherwise alter mobility.
Do RAD51C isoforms affect the Western blot pattern?
UniProt lists two RAD51C isoforms (1 and 2). Isoform 2 likely arises from alternative splicing and may run at a different apparent size or be absent depending on tissue and antibody epitope. Confirm the immunogen region covers the isoform of interest, since extra bands may reflect isoform 2 rather than degradation or nonspecific binding.
Does DNA damage change RAD51C protein levels or localization?
RAD51C is a phosphoprotein, and DNA damage induces increased nuclear accumulation, with the protein also detected in cytoplasm, perinuclear region, and mitochondria. After genotoxic treatment, expect stronger nuclear-fraction signal and possibly subtle mobility shifts from phosphorylation; compare untreated versus damage-induced lysates and fractionate nuclear and cytoplasmic pools for accurate interpretation.
What transfer method to use for RAD51C Western blot?
At ~42 kDa, RAD51C transfers efficiently using standard wet or semi-dry transfer onto 0.2 micron PVDF or nitrocellulose. With no signal peptide, propeptide, or disulfide bonds to complicate resolution, standard reducing SDS-PAGE and typical transfer times (60-90 minutes wet, or manufacturer semi-dry protocol) are sufficient for complete, uniform protein transfer.
How should RAD51C signal be normalized for quantitation?
Because RAD51C partitions between nucleus, cytoplasm, and mitochondria, and DNA damage shifts its nuclear abundance, normalize to a loading control matching the subcellular fraction analyzed rather than a single whole-cell housekeeping protein. For nuclear fractions, use a nuclear marker; for whole-cell lysates, pair with a stable total-protein stain to avoid compartment-driven quantitation errors.
What explains extra bands on a RAD51C blot?
RAD51C assembles into the ring-like BCDX2 and CX3 paralog complexes; incomplete denaturation can leave higher molecular weight complex remnants on the gel. The two annotated isoforms may also produce an additional band near 42 kDa. Rule out complex carryover by increasing SDS and reducing agent, and confirm isoform identity against the epitope location.
Boster reagents
Best RAD51C Western Blot Antibodies
BosterBio's RAD51C 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-RAD51C Antibody Picoband®
Cat # A01837-1
Real WB data
Anti-Rad51C Antibody Picoband®
Cat # PB9792
Boster's anti-RAD51C antibodies are top-performing, extensively cited Western blot reagents, rigorously validated and orthogonally cross-checked against negative-tissue controls and complementary detection methods, giving researchers confident, reproducible RAD51C detection for their experiments.
Which to pick: Two options are listed: A01837-1 and PB9792, each with a genuine RAD51C Western blot validation image. No species or additional differentiating data is provided, so either is a reasonable starting choice; compare the published blot images to your sample before deciding.
Source: BosterBio RAD51C gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry O43502.
- Human Protein Atlas. RAD51C tissue expression.