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NR1D1 / Nuclear receptor subfamily 1 group D member 1 · Western blot design guide
Design a Western Blot for NR1D1
Real validated NR1D1 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-NR1D1 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
NR1D1 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 | ~66.8 kDa | |
| Observed band | ~67 kDa | |
| Gel | 10% (catalog A01077-2) | |
| Positive control | Adrenal gland (IHC candidate; verify WB) +4 more | |
| Negative control | Adipose tissue (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated + Acetylated | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated NR1D1 Western Blot Protocols
The A01077-2 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human A549, human HepG2, human Jurkat (catalog A01077-2) |
| Gel % | 10% (catalog A01077-2) |
| Load | 30 ug; reducing conditions (catalog A01077-2) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A01077-2) |
| Membrane | nitrocellulose membrane (catalog A01077-2) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A01077-2) |
| Primary antibody | A01077-2 · 1:1000 (catalog A01077-2) |
| Primary incubation | overnight at 4°C (catalog A01077-2) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A01077-2) |
| Secondary incubation | 1.5 hour at RT (catalog A01077-2) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A01077-2) |
| Detection | ECL (catalog A01077-2) |
Section 2
What Is the Expected NR1D1 Western Blot Band Size?
NR1D1 is predicted at 66.8 kDa and observed at ~67 kDa; the cause of the small difference is not established.
| Band at ~67 kDa | Matches the observed NR1D1 band and its 66.8 kDa predicted mass |
| Band near twice the monomer size | Could reflect retained NR1D1 homodimer; band identity needs confirmation |
| ~67 kDa band enriched in a nuclear fraction | Consistent with NR1D1 nuclear localization |
| ~67 kDa band in a cytoplasmic fraction | Consistent with NR1D1 cytoplasmic localization |
💡Expected NR1D1 appearanceNR1D1 has a predicted mass of 66.8 kDa and an observed band at ~67 kDa; confirm band identity with appropriate antibody and sample controls.
| Predicted mass of 66.8 kDa | Corresponds closely to the observed ~67 kDa band |
| DNA-binding homodimer | Could appear near twice the monomer size if retained during electrophoresis |
| Phosphorylation at Ser55, Ser59 and Thr274 | May affect migration, but no visible shift is established |
| Acetylation at Lys191, Lys192, Lys400 and Lys591 | May affect migration, but no visible shift is established |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | NR1D1 may be scarce in the sampled material | Check a positive lysate and enrich the nuclear fraction |
| Band higher than expected | A retained homodimer or nonspecific binding is possible | Compare denaturing conditions and verify identity with NR1D1 depletion |
| Band lower than expected | Protein breakdown is possible; no cleavage product is specified | Use fresh lysate with protease inhibitors and verify identity |
| Multiple bands | Modification states or nonspecific binding are possible; distinct isoform bands are not supported | Compare with NR1D1 depletion and assess phosphorylation with phosphatase treatment |
| Weak or no signal | NR1D1 abundance or distribution may limit signal | Check antibody performance with a positive sample and test nuclear enrichment |
| Fragments below expected size | Protein degradation is possible | Minimize sample handling time, add protease inhibitors and compare fresh lysate |
Experiment evidence
Western blot images showing NR1D1 band
A01077-2 · NR1D1
A01077-2 · NR1D1
M01077-1 · NR1D1
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for NR1D1 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for NR1D1 in Western blot, you can use adrenal gland lysate.
Positive control: Adrenal gland (IHC candidate; verify WB)
Negative control: Adipose tissue (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: Adrenal gland has medium expression, so NR1D1 signal may be modest in whole-tissue lysate.
HPA tissue expression evidence for NR1D1
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 | Medium | Protein (IHC) | HPA → |
| Appendix | glandular cells | Medium | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Medium | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | Medium | Protein (IHC) | HPA → |
| Cerebellum | cells in granular layer | Medium | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | adipocytes | Not detected | Protein (IHC) | HPA → |
| Breast | adipocytes | Not detected | Protein (IHC) | HPA → |
| Ovary | ovarian stroma cells | Not detected | Protein (IHC) | HPA → |
| Salivary gland | glandular cells | Not detected | Protein (IHC) | HPA → |
| Smooth muscle | smooth muscle cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced NR1D1 Western Blot Tips
Deeper troubleshooting and optimisation questions for NR1D1, answered from its protein features.
How should NR1D1 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.
Do the supplied features support multiple NR1D1 isoform bands?
Isoforms · The supplied record lists one isoform and no alternative sequence. It therefore provides no isoform-based explanation for multiple bands; assess any additional bands separately.
Could phosphorylation affect NR1D1 band interpretation?
PTM · UniProt lists phosphoserines at positions 55 and 59 by GSK3-beta and phosphothreonine at 274 by CDK1. These are UniProt coordinates; check the numbering convention of any site-specific antibody. Their presence alone does not establish a resolvable band shift.
UniProt lists N6-acetyllysines at positions 191 and 192 by KAT5, plus positions 400 and 591 without a listed enzyme. Compare antibody epitope information with these UniProt coordinates when assessing whether modification might affect recognition.
UniProt lists N6-acetyllysines at positions 191 and 192 by KAT5, plus positions 400 and 591 without a listed enzyme. Compare antibody epitope information with these UniProt coordinates when assessing whether modification might affect recognition.
Does this guide establish induction of NR1D1?
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 NR1D1?
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 A01077-2 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 NR1D1 be quantified across samples?
Quantitation · The biological-rhythms annotation makes consistent collection time useful when comparing samples. Quantify the same ~67 kDa band across samples and account for the fraction analyzed, since NR1D1 has nuclear and cytoplasmic localization annotations.
Should NR1D1 run above its predicted mass?
Interpretation · The predicted mass is 66.8 kDa, and the supplied observed band is approximately 67 kDa. That agreement supports examining the ~67 kDa region. The listed modifications do not, by themselves, establish a visible shift.
UniProt annotates NR1D1 in the nucleus and cytoplasm, with dendrite and dendritic-spine localization also listed. Its note associates cytoplasmic and projection localization with OPHN1. Choose fractions according to the sample context and check fraction quality before interpreting a missing band.
Compare them with the predicted 66.8 kDa mass and the approximately 67 kDa observed band. The record lists phosphorylation and acetylation sites, but those annotations alone cannot assign an extra band or prove a mass shift. It lists no glycosylation sites or alternative sequence.
UniProt annotates NR1D1 in the nucleus and cytoplasm, with dendrite and dendritic-spine localization also listed. Its note associates cytoplasmic and projection localization with OPHN1. Choose fractions according to the sample context and check fraction quality before interpreting a missing band.
Compare them with the predicted 66.8 kDa mass and the approximately 67 kDa observed band. The record lists phosphorylation and acetylation sites, but those annotations alone cannot assign an extra band or prove a mass shift. It lists no glycosylation sites or alternative sequence.
Boster reagents
NR1D1 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-NR1D1 Antibody Picoband®
Cat # A01077-2
Real WB data
Anti-NR1D1 Rabbit Monoclonal Antibody
Cat # M01077-1
Two the supplier antibodies are listed for NR1D1, both with stated human, mouse, and rat reactivity. A01077-2 has a WB image showing a 67 kDa band in the named cell lysates. M01077-1’s image caption instead identifies KDM1B and a 55 kDa band, limiting its NR1D1 evidence.
Which to pick: Choose A01077-2 for the documented NR1D1 WB result: its caption reports a 67 kDa band in human A549, HepG2, Jurkat, HeLa, rat RH-35, and mouse NIH/3T3 lysates. M01077-1’s caption describes KDM1B, so its NR1D1 WB support is unclear.
Source: BosterBio NR1D1 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P20393.
- Human Protein Atlas. NR1D1 tissue expression.
- PMC8323274 — target-verified WB comparison
- PMC10390518 — target-verified WB comparison
- PMC9223394 — target-verified WB comparison