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NR1H4 · Western blot design guide
Design a Western Blot for NR1H4
Real validated NR1H4 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-NR1H4 WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Last reviewed: May 2026 · Scientific review: Boster Bio technical team
NR1H4 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 | ~55.9 kDa | |
| Observed band | ~56 kDa | |
| Gel | 10–12% | |
| Negative control | siRNA / KO lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated + Acetylated | |
| Caveat | Multiple splice isoforms | |
| Regulation | Bile acid metabolism | |
| Isoform | 5 isoform(s) |
Section 1
Real Curated NR1H4 Western Blot Protocols
Literature-validated Western blot parameters for NR1H4 — gel percentage, transfer, blocking, antibody incubation and detection, extracted from published methods.
| Sample / lysate | human HCCT , Lane 2: human HCCP . 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-NR1H4 antigen affinity purified polyclonal antibody (Catalog # A00835-1) 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 NR1H4 at approximately 56 kDa. The expected band size for NR1H4 is at 56 kDa |
| Gel % | 10–12% |
| 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 | 56 kDa |
Section 2
What Is the Expected NR1H4 Western Blot Band Size?
NR1H4 has a 55.9 kDa predicted backbone and runs at the same ~56 kDa observed on blots, since it lacks glycosylation, disulfide dimerization, or proteolytic cleavage to shift it.
| single band at ~56 kDa | matches the unmodified 55.9 kDa NR1H4 backbone, since there is no glycosylation, disulfide linkage, or proteolytic processing to shift it |
| one or more faint additional bands near the main ~56 kDa band | co-expression of one of the five annotated NR1H4 splice isoforms rather than a modification of the main protein |
| no band near ~112 kDa (double the monomer size) | the RXRA heterodimer is a non-covalent protein-protein interaction that dissociates under SDS-PAGE, so it does not add mass to the band |
| band present in nuclear or whole-cell lysate but weak or absent in a cytoplasm-only fraction | NR1H4 is a nuclear receptor, so signal concentrates in the nuclear compartment |
| slight fuzziness or a slightly diffuse edge around the ~56 kDa band rather than one crisp line | phosphorylation and acetylation at the annotated regulatory residues create minor charge and conformational heterogeneity |
💡Expected NR1H4 appearanceExpect a single band at approximately 56 kDa, closely matching NR1H4's predicted 55.9 kDa mass, since the protein carries no glycosylation, disulfide linkage, or signal-peptide cleavage to shift its apparent size.
| predicted mass from UniProt (55.9 kDa, 486 aa) | sets the unmodified backbone size that the observed ~56 kDa band closely tracks |
| absence of glycosylation | keeps the band sharp and near predicted size rather than smeared or shifted upward |
| absence of a signal peptide or propeptide | no proteolytic maturation step, so only the full-length species is expected, not a smaller cleaved fragment |
| alternative splicing / isoforms 1-5 | can produce additional bands of slightly different size if more than one isoform is expressed in the sample |
| phosphorylation, acetylation, and methylation at annotated regulatory residues | can cause subtle upward mobility shift or minor band fuzziness without changing the core migrating mass |
| heterodimerization with RXRA | non-covalent, so it dissociates under denaturing SDS-PAGE and does not add to the apparent band mass |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | NR1H4 is a low-abundance nuclear receptor confined to the nucleus, so whole-cell lysis without efficient nuclear extraction can under-recover it | use a nuclear extraction protocol or a lysis buffer with adequate sonication, and load a nuclear-enriched fraction |
| Multiple bands | up to five annotated NR1H4 splice isoforms can be co-expressed in the same sample | confirm the ~56 kDa band is the correct one using a knockdown or overexpression control |
| Band higher than expected | residual non-covalent RXRA heterodimer not fully dissociated, or PTM-driven mobility shift at the phosphorylated and acetylated residues | boil the sample longer in fresh reducing sample buffer with sufficient DTT or beta-mercaptoethanol to ensure full denaturation |
| Broad smear instead of sharp band | heterogeneous phosphorylation and acetylation across the six annotated modified residues | run on a higher-percentage gel for sharper resolution, or compare to a phosphatase-treated control |
| Weak or no signal | NR1H4 (bile acid receptor) is expressed mainly in liver, intestine, and kidney, and may be low or absent in the tested cell line | use a liver or intestinal tissue lysate as a positive control and increase total protein loading |
Experiment evidence
Western blot images showing NR1H4 band
A00835-1 · NR1H4
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for NR1H4 Western blot
🧪For positive controls for NR1H4 in Western blot, you can use liver tissue lysate, since the bile acid receptor (FXR) is characteristically expressed in hepatocytes.
Positive control: Liver tissue
Negative control: Non-expressing tissue (e.g. brain); or siRNA knockdown/KO line if unavailable
Loading controls: Alongside NR1H4, blot for GAPDH and β-actin, and include a total-protein stain (e.g. stain-free, Ponceau, or REVERT) to confirm equal loading.
⚠️Feasibility: HPA expression data for NR1H4 were not available, and as a nuclear receptor it is confined mostly to liver, intestine, and kidney, so a validated negative tissue cannot be confirmed from the current evidence and an siRNA/KO control is recommended to verify specificity.
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 NR1H4 Western Blot Tips
Deeper troubleshooting and optimisation questions for NR1H4, answered from its protein features.
Does the observed 56 kDa band match the predicted mass?
Yes. NR1H4's predicted mass is 55.9 kDa and the observed band runs at ~56 kDa, consistent with minimal PTM-driven shift. This close match supports specific detection; substantial deviation would suggest isoform selection, phosphorylation/acetylation/methylation adducts, or nonspecific binding rather than a genuine FXR signal.
Why might multiple NR1H4 bands appear on the blot?
NR1H4 has 5 annotated isoforms arising from alternative promoter usage and splicing, so antibodies raised against shared regions can detect several closely spaced bands near 56 kDa. Confirm which isoform your antibody's immunogen targets and compare the band pattern to tissue-specific isoform expression before labeling extra bands nonspecific.
Do post-translational modifications shift NR1H4's apparent size?
NR1H4 carries six modified residues, including phosphorylation, acetylation, methylation, and isopeptide (Ubl/SUMO) conjugation sites. Heavily modified protein pools can migrate slightly slower than the 55.9 kDa predicted mass. Treating a parallel lysate aliquot with phosphatase or deacetylase can help confirm whether a secondary band reflects a modified NR1H4 species.
What induces NR1H4 activity before probing for expression?
NR1H4 (FXR) is a bile acid-activated nuclear receptor; treating hepatic or intestinal cell models with bile acids such as chenodeoxycholic acid activates it and can increase nuclear accumulation and target-gene-driven expression. Comparing vehicle- versus bile-acid-treated lysates helps confirm antibody specificity through an expected induction response.
How should blocking be optimized for NR1H4 detection?
Because NR1H4 is a phosphoprotein with acetylation and methylation marks, use BSA rather than milk for blocking and antibody dilution to avoid phosphatase activity and casein-based interference. 5% BSA in TBST for one hour typically gives cleaner background than milk-based blocking for this nuclear receptor.
What transfer method to use for NR1H4 Western blot?
NR1H4 is a mid-size 55.9 kDa nuclear protein with no glycosylation or disulfide bonds, so standard wet or semi-dry transfer at 100V for 60-90 minutes, or overnight at low voltage, onto PVDF is sufficient. Use nuclear lysis and extraction steps, since NR1H4 localizes exclusively to the nucleus.
Which loading control suits NR1H4 quantitation?
Since NR1H4 localizes exclusively to the nucleus, normalize to a nuclear loading control such as Lamin B1 or Histone H3 rather than cytoplasmic markers like GAPDH or actin, particularly when using subcellular fractionation to enrich nuclear NR1H4 for improved sensitivity.
How to interpret unexpected bands in NR1H4 blots?
Unexpected bands can arise from the 5 NR1H4 isoforms, partial degradation, or cross-reactivity with other zinc-finger nuclear receptors sharing similar DNA-binding domains. Disease-associated variants linked to intrahepatic cholestasis may also alter migration in patient-derived samples. Validate specificity using knockdown or knockout lysate controls.
Boster reagents
Best NR1H4 Western Blot Antibodies
BosterBio's NR1H4 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-Bile Acid Receptor NR1H4 Antibody Picoband®
Cat # A00835-1
For NR1H4 Western blotting, we highlight the Boster antibody with demonstrated performance: A00835-1 includes a published SDS-PAGE Western blot image showing specific detection, giving you a concrete reference point for expected band pattern and sizing.
Which to pick: Only one NR1H4 antibody is catalogued here: A00835-1. It comes with an actual WB validation image (5-20% SDS-PAGE, 70V/90V), so use that image to compare your band pattern before troubleshooting further.
Source: BosterBio NR1H4 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q96RI1.
- Human Protein Atlas. NR1H4 tissue expression.