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- Table of Contents
RAD50 / DNA repair protein RAD50 · Western blot design guide
Design a Western Blot for RAD50
Real validated RAD50 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-RAD50 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
RAD50 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 | ~153.9 kDa | |
| Observed band | Approximately 154 kDa | |
| Gel | 8% (catalog A00347-2) | |
| Positive control | Appendix (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 + Acetylated | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 3 isoform(s) |
Section 1
Real Curated RAD50 Western Blot Protocols
The A00347-2 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human Jurkat, human MCF-7, human Hela (catalog A00347-2) |
| Gel % | 8% (catalog A00347-2) |
| Load | 30 ug; reducing conditions (catalog A00347-2) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A00347-2) |
| Membrane | nitrocellulose membrane (catalog A00347-2) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A00347-2) |
| Primary antibody | A00347-2 · 0.5 μg/mL (catalog A00347-2) |
| Primary incubation | overnight at 4°C (catalog A00347-2) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A00347-2) |
| Secondary incubation | 1.5 hour at RT (catalog A00347-2) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A00347-2) |
| Detection | ECL (catalog A00347-2) |
Section 2
What Is the Expected RAD50 Western Blot Band Size?
RAD50 is predicted at 153.9 kDa and observed at approximately 154 kDa; the supplied evidence does not establish a cause for the small difference.
| Band near 154 kDa | matches the reported RAD50 band in reducing whole-cell lysates |
| Additional bands | could reflect isoforms 1, 2, or 3; their migration is not established |
| Band above 154 kDa | could be an isoform or unrelated signal; identity requires confirmation |
| Band below 154 kDa | could be an isoform or unrelated signal; identity requires confirmation |
| Little signal in a cytoplasmic fraction | RAD50 is localized to the nucleus and chromosomes |
💡Expected RAD50 appearanceRAD50 has a predicted mass of 153.9 kDa and an empirical band at approximately 154 kDa in reducing whole-cell lysates; confirm band identity with appropriate controls.
| Predicted RAD50 mass | 153.9 kDa provides the full-length size reference |
| Splice isoform 1 | its individual apparent size is not supplied |
| Splice isoform 2 | its size relative to other isoforms is not supplied |
| Splice isoform 3 | its size relative to other isoforms is not supplied |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Nuclear RAD50 may be poorly recovered during extraction | Check nuclear recovery and probe a known positive whole-cell lysate |
| Band higher than expected | Isoform migration or band identity is unresolved | Check the size ladder and confirm identity by RAD50 depletion or a second antibody |
| Band lower than expected | Isoform migration or band identity is unresolved | Check sample integrity and confirm identity by RAD50 depletion or a second antibody |
| Multiple bands | RAD50 has three annotated isoforms, but distinct bands are unproven | Compare signals after RAD50 depletion and with a second antibody |
| Weak or no signal | Nuclear protein recovery or transfer of the 154 kDa protein may be inadequate | Check extraction, transfer, and a positive whole-cell lysate |
Experiment evidence
Western blot images showing RAD50 band
A00347-2 · RAD50
M00347 · RAD50
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for RAD50 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for RAD50 in Western blot, you can use appendix tissue lysate, which HPA scores High.
Positive control: Appendix (IHC candidate; verify WB)
Negative control: Suggested KO / knockdown lysate
Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside RAD50.
⚠️Feasibility: No supplied tissue is Not detected, so use siRNA knockdown or a KO line for a clean negative control.
HPA tissue expression evidence for RAD50
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 |
|---|---|---|---|---|
| Appendix | enterocytes | High | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | High | Protein (IHC) | HPA → |
| Breast | glandular cells | High | Protein (IHC) | HPA → |
| Bronchus | basal cells | High | Protein (IHC) | HPA → |
| Cerebellum | Purkinje cells | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | adipocytes | Low | Protein (IHC) | HPA → |
| Liver | cholangiocytes | Low | Protein (IHC) | HPA → |
| Soft tissue | fibroblasts | Low | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | Medium | Protein (IHC) | HPA → |
| Caudate | neuronal cells | Medium | Protein (IHC) | HPA → |
Section 3
Advanced RAD50 Western Blot Tips
Deeper troubleshooting and optimisation questions for RAD50, answered from its protein features.
How should RAD50 band migration be interpreted?
Band shift · Use the separately labelled calculated mass and catalog-observed evidence above. A sequence annotation does not establish an observed migration shift. Verify target identity with orthogonal controls.
Could RAD50 isoforms produce different bands?
Isoforms · UniProt lists three isoforms. Isoform 3 lacks residues 1–139; isoform 2 replaces the first five residues with an 11-residue sequence. These changes may affect band size or antibody recognition. Check which isoform your antibody detects before assigning a band.
Which RAD50 modifications matter when interpreting bands?
PTM · UniProt lists phosphoserine 635 by ATM, phosphothreonine 690, and N6-acetyllysine 959. These are UniProt coordinates; paper or antibody numbering may differ. Their presence alone does not establish a detectable band shift.
Does this guide establish induction of RAD50?
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.
What transfer method to use for RAD50 Western blot?
Transfer · RAD50 is approximately 154 kDa. Select transfer conditions suitable for a protein of this size and verify transfer efficiency near 154 kDa before interpreting a weak or absent band.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A00347-2 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 RAD50 Western blot signals be quantified?
Quantitation · Use the same sample fraction across conditions and quantify the band near 154 kDa against an appropriate loading measure. RAD50 is listed in the nucleus and on chromosomes, so changes in fractionation could change its measured signal without establishing a change in total abundance.
Why does RAD50 appear near 154 kDa?
Interpretation · The observed band near 154 kDa agrees with RAD50’s predicted mass of 153.9 kDa. The listed modifications do not establish a visible shift or explain a mass difference.
RAD50 localizes to discrete nuclear foci after treatment with genotoxic agents, and ATM phosphorylates serine 635. These features support comparing localization or site-specific phosphorylation before and after treatment; they do not establish an increase in total RAD50 abundance.
Consider the three listed isoforms, especially isoform 3, which lacks residues 1–139. UniProt also lists phosphorylation at 635 and 690 and acetylation at 959, but these features alone cannot assign an unexpected band. Check antibody recognition and compare the band with the expected approximately 154 kDa signal.
RAD50 localizes to discrete nuclear foci after treatment with genotoxic agents, and ATM phosphorylates serine 635. These features support comparing localization or site-specific phosphorylation before and after treatment; they do not establish an increase in total RAD50 abundance.
Consider the three listed isoforms, especially isoform 3, which lacks residues 1–139. UniProt also lists phosphorylation at 635 and 690 and acetylation at 959, but these features alone cannot assign an unexpected band. Check antibody recognition and compare the band with the expected approximately 154 kDa signal.
Boster reagents
RAD50 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-RAD50 Antibody Picoband®
Cat # A00347-2
Real WB data
Anti-Rad50 Monoclonal Antibody
Cat # M00347
Two the supplier anti-RAD50 antibodies have Western blot images. A00347-2 shows a band near 154 kDa in the listed human cell, rat, and mouse samples. M00347 shows RAD50 expression in Jurkat lysate; its caption provides fewer experimental details.
Which to pick: Choose A00347-2 when you want a documented blot across the listed human, rat, and mouse samples. M00347 is a monoclonal option with a Jurkat lysate blot, but the supplied evidence does not show other sample contexts for it.
Source: BosterBio RAD50 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q92878.
- Human Protein Atlas. RAD50 tissue expression.
- PMC12454627 — target-verified WB comparison
- PMC4782334 — target-verified WB comparison
- PMC8607373 — target-verified WB comparison