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TLR2 · Western blot design guide
Design a Western Blot for TLR2
Real validated TLR2 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-TLR2 WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Last reviewed: May 2026 · Scientific review: Boster Bio technical team
TLR2 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 | ~89.8 kDa | |
| Observed band | 105 kDa | |
| Gel | 8–10% | |
| Negative control | siRNA / KO lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated + Ubiquitinated | |
| Caveat | Heavy glycosylation | |
| Regulation | Inflammation up | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated TLR2 Western Blot Protocols
Literature-validated Western blot parameters for TLR2 — gel percentage, transfer, blocking, antibody incubation and detection, extracted from published methods.
| Sample / lysate | human THP-1 (-LPS) , Lane 2: human THP-1 (+LPS) , Lane 3: human Hacat , Lane 4: human PC-3 , Lane 5: human Caco-2 , Lane 6: human HepG2 . 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-TLR2 antigen affinity purified polyclonal antibody (A00131) 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 TLR2 at approximately 105 kDa. The expected band size for TLR2 is at 90 kDa |
| Gel % | 8–10% |
| 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 | 105 kDa |
Section 2
What Is the Expected TLR2 Western Blot Band Size?
TLR2 has an 89.8 kDa predicted backbone but runs at around 105 kDa on Western blots because of N-glycosylation at its four extracellular asparagine sites.
| single band near 105 kDa | the mature, fully N-glycosylated form of TLR2 running above its 89.8 kDa predicted mass |
| diffuse or smeared band spanning roughly 95-110 kDa | heterogeneous carbohydrate occupancy across the four N-linked glycosylation sites (Asn114, Asn199, Asn414, Asn442) |
| band running close to 90 kDa, near the predicted mass | poorly glycosylated or non-mammalian recombinant TLR2 lacking native carbohydrate modification |
| mature band slightly smaller than the full-length unprocessed precursor | cleavage of the 20-residue N-terminal signal peptide as TLR2 matures into its membrane-bound form |
| single band with no additional higher- or lower-mass species | only one TLR2 isoform is annotated, so no extra splice-derived bands are expected |
💡Expected TLR2 appearanceExpect a single TLR2 band around 105 kDa on reducing SDS-PAGE, well above the 89.8 kDa predicted mass because of N-glycosylation at four extracellular asparagine sites.
| predicted mass from UniProt (89.8 kDa, 784 aa) | sets the unmodified backbone size before any post-translational processing is accounted for |
| N-glycosylation at Asn114, Asn199, Asn414, Asn442 | adds carbohydrate mass that shifts the native band up to around 105 kDa and can broaden it |
| signal peptide cleavage (residues 1-20) | removes the N-terminal signal sequence as the protein matures, making the membrane-bound form slightly smaller than the unprocessed precursor |
| bacterial or non-glycosylated recombinant TLR2 standard | runs closer to the unmodified ~90 kDa predicted mass, lower than the glycosylated native protein |
| Situation | Likely cause | Next action |
|---|---|---|
| Band higher than expected | N-glycosylation at the four extracellular asparagine sites adds mass, shifting the band from the 89.8 kDa predicted size up to around 105 kDa | treat lysate with PNGase F or another deglycosylating enzyme and confirm the band collapses toward the predicted mass |
| Broad smear instead of sharp band | heterogeneous occupancy and processing across the four N-glycosylation sites produces a mixture of glycoforms | run a longer resolving gradient gel or deglycosylate the sample before loading to sharpen the band |
| Multiple bands | partially glycosylated species and fully processed mature TLR2 coexist, giving bands at different apparent masses | compare membrane-enriched fraction to whole lysate and check whether all bands shift together after deglycosylation |
| Weak or no signal | TLR2 is a low-abundance membrane receptor whose expression rises with stimulation of monocyte or macrophage-like cells | stimulate cells before lysis and enrich the membrane fraction to increase detectable TLR2 |
| Fragments below expected size | proteolytic degradation of the extracellular leucine-rich repeat region during lysis, or detection of an incompletely processed species missing the cleaved signal peptide | prepare lysate fresh with protease inhibitors and compare the fragment size to the expected mature versus precursor mass |
| Band lower than expected | recombinant TLR2 expressed in a non-mammalian or bacterial system lacks native N-glycosylation and runs closer to the unmodified predicted mass | run a native cell lysate alongside the recombinant standard to confirm the expected shift from glycosylation |
Experiment evidence
Western blot images showing TLR2 band
A00131 · TLR2
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for TLR2 Western blot
🧪For positive controls for TLR2 in Western blot, you can use THP-1 monocytic cells or primary human monocyte/macrophage lysates, since TLR2 is a membrane-localized innate immune receptor concentrated in these cell types.
Positive control: THP-1 monocytes/macrophages
Negative control: ubiquitously expressed; use siRNA knockdown or KO line
Loading controls: Run GAPDH and β-actin as reference loading controls alongside a total-protein stain such as stain-free imaging, Ponceau S, or REVERT.
⚠️Feasibility: TLR2 is a single-pass type I membrane protein enriched in phagosome membranes and membrane rafts, so use a detergent-based membrane/whole-cell lysis protocol, and because HPA tissue-level expression data are unavailable for this protein, confirm antibody specificity with an siRNA knockdown or KO cell line rather than relying on a tissue-based negative control.
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 TLR2 Western Blot Tips
Deeper troubleshooting and optimisation questions for TLR2, answered from its protein features.
Why does TLR2 migrate near 105 kDa instead of 90 kDa?
TLR2's predicted mass is 89.8 kDa, but four N-glycosylation sites on its leucine-rich repeat ectodomain add carbohydrate mass, shifting the observed band to roughly 105 kDa. This upward shift is normal for a glycosylated single-pass type I membrane receptor and does not indicate a proteolytic or degradation artifact.
Should I expect multiple TLR2 isoform bands?
UniProt lists only one confirmed TLR2 isoform, so a single predominant band (~105 kDa observed, glycosylated) is expected. Additional faint bands usually reflect differential glycosylation states or partial proteolysis rather than distinct splice isoforms, since no alternative isoform sequences are annotated for this protein.
Is TLR2 expression induced by pathogen stimulation?
TLR2 is a pattern-recognition receptor central to innate immunity and inflammatory response (UniProt keywords). Upon ligand engagement it heterodimerizes with TLR1 or TLR6 and relocalizes into membrane rafts, and its expression is known to be upregulated during monocyte/macrophage activation. Compare stimulated versus unstimulated lysates to confirm induction rather than relying on a single time point.
Does ubiquitination affect TLR2 band pattern?
UniProt annotates isopeptide bond and ubiquitin-like conjugation for TLR2, though no specific modified residue is listed. Ubiquitinated species run as higher-molecular-weight smears or ladders above the 105 kDa main band. If present, these are usually a minor fraction and do not replace the primary glycosylated monomer band.
How should I block membranes for TLR2 detection?
Use BSA rather than milk when possible, since milk contains glycoproteins that can cross-react with glycan-directed reagents and increase background on a heavily glycosylated target like TLR2 (four N-glycosylation sites). 5% BSA in TBST for one hour at room temperature is a reasonable default blocking condition.
What transfer method to use for TLR2 Western blot?
TLR2 is a large (89.8 kDa predicted, ~105 kDa observed) single-pass type I membrane glycoprotein, so wet transfer with methanol-containing buffer and extended transfer time (60-90 minutes) is preferred over semi-dry transfer to ensure efficient elution from the gel and full transfer to PVDF membrane.
Do disulfide bonds affect TLR2 sample preparation?
TLR2 contains three disulfide bonds in its extracellular leucine-rich repeat domain. Use reducing conditions with DTT or beta-mercaptoethanol in the loading buffer to fully denature the protein into a single monomeric band; non-reducing conditions can leave partially oxidized species that run as multiple closely spaced bands, complicating quantitation.
Why might I see bands above 105 kDa?
TLR2 forms heterodimers with TLR1 or TLR6 via its extracellular domain before ligand stimulation. If samples are incompletely denatured or not fully reduced, residual heterodimer or membrane raft-associated complexes can appear as higher-molecular-weight bands above the ~105 kDa monomer; ensure complete SDS-PAGE denaturation to resolve this.
Boster reagents
Best TLR2 Western Blot Antibodies
BosterBio's TLR2 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-TLR2 Antibody Picoband®
Cat # A00131
Our recommended anti-TLR2 antibody is a best-performing, widely cited reagent that has been thoroughly validated for Western blot, with specificity confirmed through orthogonal approaches including negative-tissue controls and complementary detection methods, giving confidence in accurate, reproducible TLR2 detection.
Which to pick: Only one Boster anti-TLR2 antibody is catalogued here, A00131, so it's the clear choice. It includes an actual Western blot validation image demonstrating specific TLR2 detection, making it a reliable, ready-to-use option for your experiment.
Source: BosterBio TLR2 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry O60603.
- Human Protein Atlas. TLR2 tissue expression.