This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
UCP1 / Mitochondrial brown fat uncoupling protein 1 · Western blot design guide
Design a Western Blot for UCP1
Real validated UCP1 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-UCP1 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
UCP1 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 | ~33 kDa | |
| Gel | 12–15% (standard starting point) | |
| Positive control | Brown adipose tissue (IHC candidate; verify WB) | |
| Negative control | Adipose tissue (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | — | |
| Caveat | Blocking peptide control | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated UCP1 Western Blot Protocols
The A00255 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human brain tissue lysate (catalog A00255) |
| 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 | Western blot analysis of UCP1 in human brain tissue lysate with UCP1 antibody at 1 μg/mL in (A) the absence and (B) the presence of blocking peptide (catalog A00255) |
| Primary antibody | A00255 · 1 μg/mL (catalog A00255) |
| 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 UCP1 Western Blot Band Size?
UCP1 has a predicted monomer mass of 33 kDa; its annotated features do not establish a different observed migration.
| Single band near 33 kDa | Consistent with the predicted UCP1 monomer; confirm identity with the blocking peptide. |
| Little or no band in whole-cell lysate | UCP1 resides in the mitochondrial inner membrane and may require membrane enrichment for detection. |
| Band near 66 kDa | Could reflect the proposed homodimer; a stable dimer band is not established by these features. |
| No distinct band shift after oxidation | Cys254 sulfenic acid is annotated, but a detectable mobility change is not established. |
💡Expected UCP1 appearanceUniProt predicts a 33 kDa UCP1 monomer, with no empirical band size supplied; confirm a candidate band using the antibody's blocking peptide and mitochondrial membrane enrichment.
| Predicted monomer mass | Sets a reference near 33 kDa, without establishing observed migration. |
| Proposed homodimer | Could produce a higher band near twice the monomer size if it survives sample preparation; UCP1 most probably functions as a monomer. |
| Cys254 sulfenic acid | Its effect on apparent band size is not established. |
| Multipass mitochondrial inner membrane localization | Can affect recovery during sample preparation; no specific size shift is established. |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Inner membrane UCP1 may be poorly recovered in whole-cell lysate. | Check a mitochondrial membrane fraction and membrane protein extraction. |
| Band higher than expected | A proposed oligomer or incomplete denaturation is possible; band identity is uncertain. | Compare denaturation conditions and test blocking peptide competition. |
| Band lower than expected | A fragment or unrelated band is possible; no cleavage is annotated. | Check sample integrity and blocking peptide competition. |
| Multiple bands | The supplied features do not establish multiple UCP1 isoforms or a resolvable modification pattern. | Identify which bands respond to blocking peptide and mitochondrial enrichment. |
| Weak or no signal | Insufficient recovery of mitochondrial inner membrane protein is possible. | Check membrane extraction and load a mitochondrial fraction. |
| Fragments below expected size | Sample degradation is possible; no physiological cleavage is annotated. | Check sample handling and compare freshly prepared material. |
Experiment evidence
Western blot images showing UCP1 band
A00255 · UCP1
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for UCP1 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for UCP1 in Western blot, you can use brown adipose tissue.
Positive control: Brown adipose tissue (IHC candidate; verify WB)
Negative control: Adipose tissue (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside.
⚠️Feasibility: UCP1 is an inner mitochondrial membrane protein, so mitochondrial enrichment may improve detection.
HPA tissue expression evidence for UCP1
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 |
|---|---|---|---|---|
| Brown adipose tissue | adipocytes | High | 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 → |
| Adrenal gland | glandular cells | Not detected | Protein (IHC) | HPA → |
| Appendix | glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | adipocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced UCP1 Western Blot Tips
Deeper troubleshooting and optimisation questions for UCP1, answered from its protein features.
How should UCP1 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 UCP1 isoforms explain multiple bands?
Isoforms · Only one isoform is listed, with no alternative sequence. These features do not support assigning multiple bands to annotated isoforms.
What modification is annotated for UCP1?
PTM · UniProt lists cysteine sulfenic acid (-SOH) at residue 254, using UniProt sequence numbering. This annotation alone does not show that the modification produces a visible band shift.
Does this guide establish induction of UCP1?
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 UCP1 Western blot?
Transfer · UCP1 is a predicted 33 kDa, multi-pass mitochondrial inner-membrane protein. Choose conditions that transfer and retain proteins near 33 kDa, then check transfer efficiency. The supplied features do not specify one transfer method.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the A00255 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 UCP1 bands be quantified?
Quantitation · Compare bands from equally loaded samples prepared and measured consistently. Because UCP1 resides in the mitochondrial inner membrane, differences in mitochondrial content or recovery can affect the signal independently of UCP1 abundance.
Should UCP1 run at its predicted 33 kDa?
Interpretation · The predicted mass is 33 kDa, but no observed band position is supplied. Use 33 kDa as a reference, not a guaranteed migration position; the listed features do not establish a band shift.
Do not assign an unexpected band to an isoform or oxidation based on these features alone. UCP1 is most probably monomeric, although larger assemblies have been suggested; that uncertainty does not identify a band. Confirm band identity experimentally.
Do not assign an unexpected band to an isoform or oxidation based on these features alone. UCP1 is most probably monomeric, although larger assemblies have been suggested; that uncertainty does not identify a band. Confirm band identity experimentally.
Boster reagents
UCP1 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-UCP1 Antibody
Cat # A00255
A00255 is listed as reactive with human, mouse, and rat UCP1. Its Western blot image uses human brain tissue lysate at 1 μg/mL, with and without blocking peptide. The supplied evidence does not show mouse or rat blots.
Which to pick: A00255 is the only listed option. Its Western blot image shows human brain tissue lysate with a blocking-peptide comparison; mouse and rat are listed as reactive, but no blots for those species are supplied.
Source: BosterBio UCP1 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P25874.
- Human Protein Atlas. UCP1 tissue expression.
- PMC11718246 — target-verified WB comparison
- PMC12491148 — target-verified WB comparison
- PMC6857924 — target-verified WB comparison