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ATR · Western blot design guide
Design a Western Blot for ATR
Real validated ATR Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-ATR WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Last reviewed: May 2026 · Scientific review: Boster Bio technical team
ATR 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 | ~301.4 kDa | |
| Observed band | ~301 kDa | |
| Gel | 4–12% gradient | |
| Negative control | siRNA / KO lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated | |
| Caveat | Alternative splicing isoforms | |
| Regulation | Upregulated (immune signature) | |
| Isoform | 3 isoform(s) |
Section 1
Real Curated ATR Western Blot Protocols
Literature-validated Western blot parameters for ATR — gel percentage, transfer, blocking, antibody incubation and detection, extracted from published methods.
| Sample / lysate | human Hela , Lane 2: human Daudi . 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-ATR antigen affinity purified polyclonal antibody (Catalog # A00262-3) 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 ATR at approximately 301 kDa. The expected band size for ATR is at 301 kDa |
| Gel % | 4–12% gradient |
| 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 | 301 kDa |
Section 2
What Is the Expected ATR Western Blot Band Size?
ATR has a 301.4 kDa predicted mass and runs at the empirically observed ~301 kDa, with heavy phosphorylation as the main source of minor mobility shifts.
| single band at ~301 kDa | matches the 301.4 kDa predicted mass of full-length ATR with no major shifting modification |
| band sitting slightly above the unmodified 301 kDa backbone | multiple phosphorylation sites, including autophosphorylated Thr1989, add mass and can retard migration slightly |
| faint extra bands distinct from the main ~301 kDa band | ATR has three annotated splice isoforms that can migrate at different apparent sizes if co-expressed |
| no doubling of band size under standard reducing SDS-PAGE | ATR-ATRIP is a non-covalent heterodimer, not disulfide-linked, so it dissociates under reducing and denaturing conditions |
| band still detected in whole-cell lysate rather than only a nuclear fraction | ATR is nuclear and chromosome-associated, but whole-cell lysis captures the nuclear compartment along with the rest of the cell |
💡Expected ATR appearanceATR is expected to run as a single band at the empirically observed ~301 kDa, closely matching its 301.4 kDa predicted mass with no glycosylation or disulfide-linked shift.
| Predicted mass (301.4 kDa) | sets the baseline expected migration for this very large kinase, requiring low-percentage gels and extended run and transfer times |
| Phosphorylation at Ser428, Ser435, Ser436, and autocatalytic Thr1989 | can cause the observed band to run slightly higher than the unmodified 301 kDa backbone |
| Alternative splice isoforms 1, 2, and 3 | isoform 2 or 3 may appear as additional bands of somewhat different apparent size relative to the canonical isoform 1 band |
| ATR-ATRIP heterodimer formation | is a non-covalent association that dissociates under reducing, denaturing SDS-PAGE, so it does not add mass to the detected band |
| Absence of signal peptide or propeptide cleavage | means no smaller mature or cleaved form is expected; the full-length translated product is what is detected |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | ATR is a large, low-abundance nuclear and chromatin-bound protein that can be under-extracted by insufficient lysis | use a nuclear extraction or high-salt lysis buffer with sonication and load a higher amount of total protein |
| Band higher than expected | extensive phosphorylation, including autophosphorylation at Thr1989, retards migration relative to the unmodified backbone | treat a parallel lysate aliquot with lambda phosphatase and compare mobility to confirm a phospho-dependent shift |
| Weak or no signal | ATR is both very large and typically low in cellular abundance, which reduces transfer efficiency and detectable signal | use a low-percentage gel, extend wet transfer time, and increase total protein loaded per lane |
| Multiple bands | the three annotated splice isoforms of ATR can each produce a band of somewhat different apparent size | check which isoform the antibody epitope falls within and confirm band identity against isoform-specific sequence differences |
| Fragments below expected size | a protein of this size is prone to proteolytic degradation during lysis if protease activity is not adequately controlled | keep samples cold, add fresh protease inhibitor cocktail, and minimize freeze-thaw cycles before loading |
Experiment evidence
Western blot images showing ATR band
A00262-3 · ATR
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for ATR Western blot
🧪For positive controls for ATR in Western blot, you can use HeLa cell lysate, since ATR is a ubiquitously expressed nuclear checkpoint kinase present in essentially all proliferating cell lines.
Positive control: HeLa cells
Negative control: ubiquitously expressed; use siRNA knockdown or KO line
Loading controls: Run GAPDH and β-actin as housekeeping loading controls alongside a total-protein stain (stain-free gel, Ponceau S, or REVERT) to normalize for the large (~301 kDa) ATR band.
⚠️Feasibility: Because ATR is a large, low-abundance nuclear kinase expressed across nearly all tissues, no tissue serves as a true negative control, so validation should rely on siRNA knockdown or a CRISPR knockout line rather than tissue selection.
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 ATR Western Blot Tips
Deeper troubleshooting and optimisation questions for ATR, answered from its protein features.
Why does ATR run at its predicted 301 kDa mass?
ATR has no signal peptide, propeptide, glycosylation, or disulfide bonds, so its migration reflects the unmodified 301.4 kDa polypeptide. The observed ~301 kDa band matches prediction closely, indicating minimal post-translational mass addition; any consistent shift more likely reflects incomplete transfer or gel percentage than true PTM-driven mobility change for this protein.
Could isoform 2 or 3 cause extra bands?
UniProt lists three ATR isoforms (1-3) from alternative splicing. If the antibody epitope falls outside a spliced region, only isoform 1 (canonical, 301 kDa) may be detected; if it targets a shared region, additional bands near but not at 301 kDa can appear. Confirm epitope location against isoform sequences before treating extra bands as nonspecific.
Does DNA damage increase ATR phosphorylation signal?
ATR carries four annotated modified residues and is activated by phosphorylation during the DNA damage and replication stress response as part of the ATR-ATRIP complex. Treating cells with agents inducing replication stress (e.g., hydroxyurea) or DNA damage (e.g., UV) increases ATR phosphorylation, useful for validating phospho-specific ATR antibodies; total ATR protein levels change less than its phosphorylated fraction.
What blocking buffer avoids phospho-ATR signal loss?
Because ATR activation is phosphorylation-dependent, use BSA rather than milk when probing with phospho-specific ATR antibodies, as casein phosphoproteins in milk can cause background or reduce phospho-epitope detection. For total ATR detection, standard milk blocking is acceptable since no glycosylation is annotated.
What transfer method to use for ATR Western blot?
At 301 kDa, ATR requires low-percentage (5-6%) SDS-PAGE and extended wet transfer (overnight at low voltage/4°C, or high-voltage tank transfer) onto PVDF to ensure efficient large-protein migration out of the gel; standard rapid semi-dry transfer often under-transfers proteins this size, causing weak or absent signal.
Which loading control suits nuclear ATR normalization?
ATR is a nuclear and chromosome-associated protein, so normalize to a nuclear marker such as Lamin B1 or Histone H3 rather than cytoplasmic controls like GAPDH, particularly when using nuclear/cytoplasmic fractionation to enrich chromatin-bound ATR before blotting.
Are lower molecular weight ATR bands degradation?
Given ATR's large size (301 kDa) and lack of stabilizing disulfide bonds, lower-MW bands commonly reflect proteolytic degradation during lysis rather than distinct isoforms. Use fresh lysates with protease inhibitors, keep samples cold, and avoid repeated freeze-thaw to minimize fragment bands.
Boster reagents
Best ATR Western Blot Antibodies
BosterBio's ATR 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-ATR Antibody Picoband®
Cat # A00262-3
Boster's anti-ATR antibody A00262-3 is a best-performing, thoroughly validated reagent for ATR Western blotting, widely cited in the literature and orthogonally cross-validated against negative tissue and complementary detection methods to ensure specificity and reliable results.
Which to pick: Only one ATR antibody is catalogued, A00262-3, which includes an actual Western blot validation image—making it the clear, ready-to-use choice for ATR WB experiments without needing to compare alternatives.
Source: BosterBio ATR gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q13535.
- Human Protein Atlas. ATR tissue expression.