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RAD17 / Cell cycle checkpoint protein RAD17 · Western blot design guide
Design a Western Blot for RAD17
Source-linked RAD17 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-RAD17 WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Evidence assembled October 2026 · For research use; verify linked source records and product datasheet before use
RAD17 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 | ~77.1 kDa | |
| Observed band | 85 kDa | |
| Gel | 12–15% (standard starting point) | |
| Positive control | Bone marrow (total-target IHC; phospho state unverified) +4 more | |
| Negative control | Cervix (total-target IHC; phospho state unverified) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated | |
| Caveat | Activation-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 4 isoform(s) |
Section 1
Source-Linked RAD17 Western Blot Protocol Options
The P02159-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | (1) HeLa cell treated with CA lysate; (2) HeLa cell lysate (catalog P02159-1) |
| 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 | 5% BSA in TBST (standard starting point) |
| Primary antibody | P02159-1; 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 RAD17 Western Blot Band Size?
RAD17 is predicted at 77.1 kDa and observed at 85 kDa; the cause is not established, and the lead antibody recognizes phospho-Ser656 RAD17.
| Band near 85 kDa | Empirically observed RAD17 band; confirm identity with appropriate controls |
| Band near 77.1 kDa | Near the predicted RAD17 mass, but identity requires confirmation |
| Several distinct bands | Could include RAD17 isoforms 1, 2, 3, and 4; their separation is unestablished |
| Band detected by phospho-S656 antibody | Consistent with recognition of Ser656-phosphorylated RAD17, subject to specificity controls |
💡Expected RAD17 appearanceRAD17 is predicted at 77.1 kDa and has an empirical band at 85 kDa; the difference is unexplained, so verify band identity, especially with the phospho-S656-specific antibody.
| Predicted RAD17 molecular weight | 77.1 kDa from the supplied sequence; apparent migration can differ |
| RAD17 isoform 1 | Its individual mass and migration are not supplied |
| RAD17 isoform 2 | Its size relative to isoform 1 is not established |
| RAD17 isoform 3 | Its size relative to other isoforms is not established |
| RAD17 isoform 4 | Its size relative to other isoforms is not established |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Nuclear or chromosome-associated RAD17 may be poorly recovered | Check nuclear extraction and a total-RAD17 positive control |
| Band higher than expected | The empirical 85 kDa band exceeds the 77.1 kDa predicted mass for an unestablished reason | Confirm identity using RAD17 depletion or an independent antibody |
| Band lower than expected | An alternative isoform is possible, but isoform masses are unknown | Confirm RAD17 identity with depletion and an isoform-aware assay |
| Multiple bands | RAD17 has four named isoforms, but their migration has not been established | Compare bands after RAD17 depletion and with an independent antibody |
| Weak or no signal | A phospho-S656-specific antibody depends on recognition of the phosphorylated target state | Compare a suitable phospho-positive control with total RAD17 |
Experiment evidence
Western blot images showing RAD17 band
P02159-1 · RAD17
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for RAD17 Western blot
🧪HPA-IHC candidate guidance (verify in WB): HPA tissue expression does not establish phosphorylation at the selected antibody epitope. Verify activation-state controls for the phospho-specific lead. HPA-IHC candidate guidance (verify in WB): For positive controls for RAD17 in Western blot, you can use bone marrow lysate, which HPA rates as highly positive.
Positive control: Bone marrow (total-target IHC; phospho state unverified)
Negative control: Cervix (total-target IHC; phospho state unverified)
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside RAD17.
⚠️Feasibility: HPA lists cervix as not detected, making a tissue negative control feasible, but confirm the result in your lysate.
HPA tissue expression evidence for RAD17
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 |
|---|---|---|---|---|
| Bone marrow | hematopoietic cells | High | Protein (IHC) | HPA → |
| Lung | macrophages | High | Protein (IHC) | HPA → |
| Placenta | trophoblastic cells | High | Protein (IHC) | HPA → |
| Testis | pachytene spermatocytes | High | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | Medium | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Cervix | glandular cells | Not detected | Protein (IHC) | HPA → |
| Oral mucosa | squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Parathyroid gland | glandular cells | Not detected | Protein (IHC) | HPA → |
| Seminal vesicle | glandular cells | Not detected | Protein (IHC) | HPA → |
| Soft tissue | fibroblasts | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced RAD17 Western Blot Tips
Deeper troubleshooting and optimisation questions for RAD17, answered from its protein features.
How should RAD17 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.
Which RAD17 isoforms could affect band interpretation?
Isoforms · UniProt lists isoforms 1–4. Compared with the canonical sequence, isoform 3 lacks residues 1–176; isoform 4 lacks 1–97 and replaces 98–99 (ET) with MN; isoform 2 replaces 1–14 (MSKTFLRPKVSSTK) with MNQ. Antibodies against the missing N-terminal regions may miss some isoforms.
Which RAD17 phosphorylation sites matter when interpreting bands?
PTM · In UniProt canonical numbering, RAD17 has phosphothreonine at 55 and 633, and phosphoserine at 71, 86, 359, 646 and 656. ATM modifies 633; ATR and ATM modify 646 and 656. These sites do not, by themselves, establish a visible band shift. Check whether an antibody’s site numbering follows the same sequence convention.
Does this guide establish induction of RAD17?
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 RAD17?
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 P02159-1 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 RAD17 be quantified across DNA damage conditions?
Quantitation · Compare the same sample fraction across conditions, since phosphorylated RAD17 can redistribute to nuclear foci after DNA damage. Quantify the intended RAD17 band consistently; if assessing a phosphosite, distinguish that signal from total RAD17. The listed sites alone do not establish a change in total protein.
Why might RAD17 appear at 85 kDa instead of 77.1 kDa?
Interpretation · 77.1 kDa is the predicted mass; 85 kDa is the reported apparent band. RAD17 has phosphorylation sites and alternative isoforms, but these features alone do not establish the cause of the difference or demonstrate a visible shift.
UniProt reports that phosphorylated RAD17 redistributes to discrete nuclear foci upon DNA damage. A change in a nuclear or chromosome-associated fraction could therefore reflect redistribution. The supplied features do not establish that DNA damage increases total RAD17 abundance.
Consider the four annotated isoforms and whether the antibody recognizes their differing N-terminal sequences. Compare additional bands with the reported 85 kDa apparent band and the 77.1 kDa predicted canonical mass. The supplied features list no glycosylation sites, signal peptide or propeptide; they do not identify the additional bands.
UniProt reports that phosphorylated RAD17 redistributes to discrete nuclear foci upon DNA damage. A change in a nuclear or chromosome-associated fraction could therefore reflect redistribution. The supplied features do not establish that DNA damage increases total RAD17 abundance.
Consider the four annotated isoforms and whether the antibody recognizes their differing N-terminal sequences. Compare additional bands with the reported 85 kDa apparent band and the 77.1 kDa predicted canonical mass. The supplied features list no glycosylation sites, signal peptide or propeptide; they do not identify the additional bands.
Boster reagents
RAD17 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-Phospho-Rad17 (S656) Rabbit Monoclonal Antibody
Cat # P02159-1
P02159-1 is a rabbit monoclonal antibody against phospho-Rad17 (S656) with reported human reactivity. Its Western blot image shows CA-treated HeLa cell lysate and HeLa cell lysate; the supplied evidence is limited to these samples.
Which to pick: P02159-1 is the only listed RAD17 antibody. Choose it when measuring phospho-Rad17 at S656 in a human sample; its supplied Western blot image uses HeLa lysates.
Source: BosterBio RAD17 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry O75943.
- Human Protein Atlas. RAD17 tissue expression.
- PMC9340372 — target-verified WB comparison