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- Table of Contents
RAD51B / DNA repair protein RAD51 homolog 2 · Western blot design guide
Design a Western Blot for RAD51B
Source-linked RAD51B Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-RAD51B 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
RAD51B Western Blot Experimental Design Guide
Expected bands, source-linked protocol options, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before running
| Expected band | ~42.2 kDa | |
| Gel | 12–15% (standard starting point) | |
| Positive control | Adrenal gland (IHC candidate; verify WB) +4 more | |
| Negative control | Suggested KO / knockdown lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 5 isoform(s) |
Section 1
Source-Linked RAD51B Western Blot Protocol Options
The A20019 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | lysates from COS7 cells, (catalog A20019) |
| Gel % | 12–15% (standard starting point) |
| Load | 20–30 µg total protein per lane; optimize for abundance (standard starting point) |
| Transfer | Standard semi-dry transfer; verify efficiency (standard starting point) |
| Membrane | 0.45 µm PVDF (standard starting point) |
| Blocking | the synthesized peptide (catalog A20019) |
| Primary antibody | A20019; use the WB datasheet starting dilution (standard starting point) |
| Primary incubation | Overnight at 4 °C (standard starting point) |
| Secondary antibody | Species-matched HRP conjugate at validated dilution (standard starting point) |
| Secondary incubation | 1 h at room temperature (standard starting point) |
| Wash | 3 × 5 min in TBST (standard starting point) |
| Detection | ECL; bracket exposures to avoid saturation (standard starting point) |
Section 2
What Is the Expected RAD51B Western Blot Band Size?
RAD51B is predicted at 42.2 kDa; five isoforms could affect band patterns, but distinct migration has not been demonstrated.
| Band near 42.2 kDa | Consistent with the predicted RAD51B size; confirm identity with controls |
| Two bands at different positions | Could reflect splice isoforms; their migration is not established |
| Several bands at different positions | Could reflect the five annotated isoforms; confirm each band’s identity |
| Stronger band in a nuclear fraction | Consistent with RAD51B’s nuclear location |
💡Expected RAD51B appearanceRAD51B has a predicted mass of 42.2 kDa; no empirical band size or isoform-specific migration is supplied, so confirm any candidate band with ordinary identity controls.
| UniProt predicted mass | Places the predicted protein near 42.2 kDa |
| Splice isoform 1 | Its individual mass and migration are not supplied |
| Splice isoform 2 | May differ in size; its migration is not established |
| Splice isoform 3 | May differ in size; its migration is not established |
| Splice isoform 4 | May differ in size; its migration is not established |
| Splice isoform 5 | May differ in size; its migration is not established |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Nuclear RAD51B may be dilute in whole-cell lysate | Check a nuclear fraction and a positive control |
| Multiple bands | RAD51B has five annotated splice isoforms, but their migration is unknown | Use knockdown and isoform-aware controls to identify bands |
| Band higher than expected | No listed modification establishes a higher apparent mass | Check band identity with knockdown and an independent antibody |
| Band lower than expected | An isoform is possible, but no isoform-specific mass is supplied | Check band identity and antibody epitope coverage |
| Weak or no signal | Nuclear RAD51B may be underrepresented in the sample | Verify loading and enrich the nuclear fraction |
Experiment evidence
Western blot images showing RAD51B band
A20019 · RAD51B
A02658-2 · RAD51B
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for RAD51B Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for RAD51B in Western blot, you can use adrenal gland lysate, which has high HPA expression.
Positive control: Adrenal gland (IHC candidate; verify WB)
Negative control: Suggested KO / knockdown lysate
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: No supplied tissue is HPA Not detected, so use siRNA knockdown or a KO line for a negative control.
HPA tissue expression evidence for RAD51B
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 |
|---|---|---|---|---|
| Adrenal gland | glandular cells | High | Protein (IHC) | HPA → |
| Appendix | glandular cells | High | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | High | Protein (IHC) | HPA → |
| Breast | glandular cells | High | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | adipocytes | Medium | Protein (IHC) | HPA → |
| Heart muscle | cardiomyocytes | Medium | Protein (IHC) | HPA → |
| Prostate | glandular cells | Medium | Protein (IHC) | HPA → |
| Vagina | squamous epithelial cells | Medium | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | High | Protein (IHC) | HPA → |
Section 3
Advanced RAD51B Western Blot Tips
Deeper troubleshooting and optimisation questions for RAD51B, answered from its protein features.
Where should the canonical RAD51B band appear?
Band shift · The canonical 384-residue sequence has a predicted mass of 42.2 kDa. No observed band position is supplied, so use 42.2 kDa as a reference, not a guaranteed migration position.
Could RAD51B isoforms produce different bands?
Isoforms · Five isoforms are listed. Isoform 5 lacks residues 1–119; isoforms 2, 3, 4 and 5 have changes near the C-terminus. These sequence differences could affect apparent bands, but the features do not establish which isoforms are expressed or visible.
Check whether the epitope overlaps residues 1–119, absent from isoform 5, or the altered C-terminal region around residues 346–384. An antibody against either region may recognize the isoforms differently. Coordinates here follow the supplied UniProt canonical sequence.
Check whether the epitope overlaps residues 1–119, absent from isoform 5, or the altered C-terminal region around residues 346–384. An antibody against either region may recognize the isoforms differently. Coordinates here follow the supplied UniProt canonical sequence.
Can phosphorylation explain a shifted RAD51B band?
PTM · UniProt labels RAD51B a phosphoprotein but lists no modified residue or phosphorylation site in the supplied features. That keyword alone cannot establish a visible shift or explain a measured mass difference.
Does this guide establish induction of RAD51B?
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.
How should transfer be checked for RAD51B?
Transfer · Standard workflow guidance: verify transfer efficiency for the intended target size before interpreting a weak signal. Use total-protein assessment and optimize transfer for the membrane, gel and apparatus; the labelled catalog values take precedence.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A20019 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 RAD51B bands be quantified across samples?
Quantitation · Keep the antibody epitope in mind when comparing bands: the five listed isoforms differ at the N-terminus or C-terminus. Quantify the same defined band or band set across samples, and report which bands were included.
How should unexpected RAD51B bands be interpreted?
Interpretation · Compare them with the 42.2 kDa canonical prediction and the listed isoform sequence changes. A band at another position is not, by itself, evidence of an isoform or modification; the supplied features include no empirical band position or mapped modified residue.
Boster reagents
RAD51B 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-RAD51L1 RAD51B Antibody
Cat # A20019
Real WB data
Anti-RAD51L1/RAD51B Antibody Picoband®
Cat # A02658-2
Two the supplier anti-RAD51B antibodies have WB images: A20019 in COS7 lysate with a peptide-blocked lane, and A02658-2 in three human cell lysates with a reported ~42 kDa band. These examples do not establish performance across all listed reactive species or sample types.
Which to pick: For human cell lysates, A02658-2 has documented WB results in Jurkat, HaCaT and U2OS samples. A20019 lists human, monkey and mouse reactivity, with a COS7 WB image and peptide-blocked lane; the supplied evidence does not show WB results for every listed species.
Source: BosterBio RAD51B gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry O15315.
- Human Protein Atlas. RAD51B tissue expression.
- PMC4965152 — target-verified WB comparison