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FFAR1 · Western blot design guide
Design a Western Blot for FFAR1
Real validated FFAR1 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-FFAR1 WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Last reviewed: May 2026 · Scientific review: Boster Bio technical team
FFAR1 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 | ~31.5 kDa | |
| Observed band | 31 kDa | |
| Gel | 10–12% | |
| Negative control | siRNA / KO lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated | |
| Caveat | Glycosylation may alter migration | |
| Regulation | IL-6-induced | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated FFAR1 Western Blot Protocols
Literature-validated Western blot parameters for FFAR1 — gel percentage, transfer, blocking, antibody incubation and detection, extracted from published methods.
| Sample / lysate | human PANC-1 . After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA 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-GPCR GPR40 antigen affinity purified polyclonal antibody (Catalog # A01349-2) 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 1:10000 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 GPCR GPR40 at approximately 31KD. The expected band size for GPCR GPR40 is at 31KD |
| Gel % | 10–12% |
| Load | 50ug |
| 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, 1:10000 |
| Wash | TBS-0.1% Tween, 3 × 5 min |
| Detection | ECL |
| Exposure / imaging | Tanon 5200 |
| Observed band | 31 kDa |
Section 2
What Is the Expected FFAR1 Western Blot Band Size?
FFAR1 has a 31.5 kDa predicted mass and is observed at 31 kDa, essentially matching prediction since its single Asn155 glycan adds little detectable shift.
| Single band at approximately 31 kDa | Matches the FFAR1 monomer, close to its 31.5 kDa predicted mass with little apparent shift from modifications |
| Band running slightly above the 31 kDa core, sometimes diffuse | Reflects the single N-linked glycan at Asn155 adding modest, heterogeneous mass |
| Broad or fuzzy band rather than a crisp line | Typical of a seven-transmembrane GPCR whose hydrophobic helices bind SDS unevenly and migrate anomalously |
| Band position shifts between reducing and non-reducing sample prep | The Cys79-Cys170 intrachain disulfide holds the extracellular loop folded, so an unreduced sample runs more compactly than a reduced one |
| No band, or very faint band, in standard whole-cell lysate | FFAR1 is a multi-pass plasma membrane protein that needs adequate detergent solubilization to be extracted and detected |
💡Expected FFAR1 appearanceFFAR1 is expected as a single band around 31 kDa, consistent with its 31.5 kDa predicted mass and the modest mass contribution of its lone Asn155 glycan.
| Predicted mass from UniProt (31.5 kDa) | Sets the core backbone size, closely matched by the empirically observed 31 kDa band |
| N-glycosylation at Asn155 | Adds a modest, heterogeneous mass that can broaden or slightly elevate the band above the unmodified core |
| Intrachain disulfide bond (Cys79-Cys170) | Stabilizes the folded extracellular loop, so incompletely reduced samples can migrate faster or as a distinct species compared with fully reduced samples |
| Multi-pass membrane topology (seven-transmembrane GPCR) | Hydrophobic transmembrane helices bind SDS unevenly, often producing anomalous or smeared migration relative to true mass |
| Bacterial or non-glycosylated recombinant standard | Lacking the Asn155 glycan, runs closer to or at the bare 31.5 kDa backbone, at or below the native band |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Standard whole-cell lysis may not adequately solubilize this multi-pass membrane receptor | Use a membrane-protein lysis buffer with sufficient detergent such as Triton X-100 or DDM and load more total protein |
| Band higher than expected | Heterogeneous N-glycosylation at Asn155 adds extra apparent mass | Treat lysate with PNGase F to deglycosylate and collapse the band toward the 31.5 kDa core |
| Band lower than expected | Membrane proteins can be partially degraded or fail to fully retain SDS, causing faster than expected migration | Add fresh protease inhibitors during lysis and avoid prolonged sample handling before loading |
| Broad smear instead of sharp band | Glycan heterogeneity and incomplete, uneven SDS binding to the transmembrane helices | Deglycosylate with PNGase F and avoid boiling the sample, using moderate heat instead to limit membrane protein aggregation |
| Multiple bands | Incomplete reduction leaves a disulfide-stabilized conformer alongside the fully reduced monomer, or the antibody shows nonspecific binding | Ensure complete reduction with fresh DTT or beta-mercaptoethanol and confirm specificity with a knockdown or knockout control |
| Weak or no signal | Low native abundance and inefficient extraction of this membrane receptor from cell lysate | Enrich for membrane fractions before loading and increase total protein loaded per lane |
Experiment evidence
Western blot images showing FFAR1 band
A01349-2 · FFAR1
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for FFAR1 Western blot
🧪For positive controls for FFAR1 in Western blot, you can use a recombinant FFAR1-overexpressing cell lysate, since Human Protein Atlas data do not report a confirmed endogenous-positive tissue or cell line for this protein.
Positive control: FFAR1-overexpressing cell lysate
Negative control: ubiquitously expressed; use siRNA knockdown or KO line
Loading controls: Run GAPDH and β-actin alongside a total-protein stain (e.g., stain-free gel, Ponceau S, or REVERT) as loading controls.
⚠️Feasibility: As a multi-pass membrane GPCR with no HPA-confirmed positive tissue, native controls are hard to source, so an overexpression lysate paired with siRNA/KO validation is the more feasible option.
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 FFAR1 Western Blot Tips
Deeper troubleshooting and optimisation questions for FFAR1, answered from its protein features.
Why does FFAR1 run near its predicted 31 kDa mass?
FFAR1 has only one N-glycosylation site and no other modified residues, so glycosylation adds minimal mass. The 31 kDa observed band closely matches the 31.5 kDa predicted mass, indicating little post-translational processing shifts migration on SDS-PAGE.
Could the single glycosylation site alter FFAR1 band size?
FFAR1 carries one predicted N-glycosylation site. Because it is a single site rather than multiple, any size increase from glycosylation is small and may not resolve as a separate band; expect a single sharp band near 31 kDa rather than a glycosylation ladder.
Should isoform variation be considered for FFAR1 blots?
UniProt lists only one FFAR1 isoform, so multiple bands from alternative splicing are not expected. A single specific band near 31 kDa should represent the full-length receptor; additional bands likely reflect aggregation or nonspecific binding rather than isoforms.
How should blocking be optimized for FFAR1 detection?
FFAR1 is a lipid-binding, multi-pass membrane receptor, so membrane preparations carry residual lipids that increase background. Use BSA-based blocking rather than milk to reduce nonspecific lipid and phosphoprotein interactions, and include detergent (e.g., 0.1% Tween-20) in wash buffers to clear membrane debris.
What transfer method to use for FFAR1 Western blot?
FFAR1 is a multi-pass transmembrane GPCR with a disulfide bond stabilizing its structure, making it prone to poor elution and aggregation. Use wet transfer with PVDF membrane, low methanol (10%), and extended transfer time to improve recovery of this hydrophobic receptor.
How to normalize quantitation of FFAR1 signal?
Since FFAR1 is a cell membrane, multi-pass membrane protein, normalize to a membrane-resident loading control such as Na+/K+-ATPase rather than cytosolic GAPDH, ensuring equal loading of the membrane fraction being probed.
What explains unexpected higher molecular weight FFAR1 bands?
FFAR1 contains one disulfide bond and is a multi-pass membrane protein prone to aggregation if incompletely denatured. Higher MW bands likely represent disulfide-linked dimers or SDS-resistant aggregates; adding fresh reducing agent and heating lysates longer before loading can help resolve the monomeric 31 kDa band.
Does FFAR1 expression need induction before blotting?
UniProt localizes FFAR1 to the cell membrane without induction/expression feature annotations. As a fatty-acid receptor, endogenous expression is naturally limited to specific tissues (e.g., pancreatic beta cells), so confirm sufficient basal expression in the chosen sample rather than expecting an inducible increase.
Boster reagents
Best FFAR1 Western Blot Antibodies
BosterBio's FFAR1 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-GPCR GPR40/FFAR1 Antibody Picoband®
Cat # A01349-2
For FFAR1 (GPR40) Western blot, Boster offers antibody A01349-2, supported by a documented WB image showing detection of GPCR GPR40 under standard SDS-PAGE conditions — a practical starting point for your assay.
Which to pick: Only one FFAR1 antibody is catalogued here: A01349-2. It includes an actual WB validation image, so it's the default choice; confirm species reactivity and lysate type match your sample before ordering.
Source: BosterBio FFAR1 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry O14842.
- Human Protein Atlas. FFAR1 tissue expression.