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TRPM8 · Western blot design guide
Design a Western Blot for TRPM8
Real validated TRPM8 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-TRPM8 WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Last reviewed: May 2026 · Scientific review: Boster Bio technical team
TRPM8 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 | ~127.7 kDa | |
| Observed band | ~127 kDa | |
| Gel | 8–10% | |
| Negative control | siRNA / KO lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated | |
| Caveat | N-glycosylation | |
| Regulation | LPS-suppressed | |
| Isoform | 4 isoform(s) |
Section 1
Real Curated TRPM8 Western Blot Protocols
Literature-validated Western blot parameters for TRPM8 — gel percentage, transfer, blocking, antibody incubation and detection, extracted from published methods.
| Sample / lysate | HELA , Lane 2: 22RV1 , Lane 3: SW620 , Lane 4: A549 , Lane 5: A431 . 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-TRPM8 antigen affinity purified polyclonal antibody (Catalog # PB9837) 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 TRPM8 at approximately 127 kDa. The expected band size for TRPM8 is at 127 kDa |
| Gel % | 8–10% |
| Load | 40 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 | 127 kDa |
Section 2
What Is the Expected TRPM8 Western Blot Band Size?
TRPM8 has a 127.7 kDa predicted backbone and runs at the empirically observed ~127 kDa monomer, with minor shifts possible from its single Asn934 N-glycan and its non-covalent homotetramer assembly.
| Single band at ~127 kDa | Matches the full-length TRPM8 monomer; there is no signal peptide or propeptide to trim off, so the observed band reflects the intact predicted mass. |
| Band sitting slightly above the unmodified 127.7 kDa core | The single N-linked complex-type glycan at Asn934 adds a small mass increment to the mature channel. |
| High-molecular-weight band or smear near the top of the gel | TRPM8 assembles as a non-covalent homotetramer; incompletely denatured/reduced samples can retain tetrameric or partial oligomeric complexes. |
| Multiple bands at different apparent masses | Four annotated splice isoforms (1-4), including isoforms that interact with the isoform-1 C-terminus, can contribute additional bands beyond the primary monomer. |
| Fuzzy edge or minor doublet around the main band | Microheterogeneity in occupancy/processing of the single glycosylation site at Asn934 can slightly blur the primary band. |
💡Expected TRPM8 appearanceExpect a single dominant TRPM8 band at the empirically observed ~127 kDa, matching the 127.7 kDa predicted monomer mass with only a minor upward shift from the single N-glycan at Asn934.
| Predicted mass (127.7 kDa) | Sets the baseline monomer size, consistent with the empirically observed ~127 kDa band. |
| N-glycosylation at Asn934 (complex-type) | Adds a small mass increment and can slightly broaden the band relative to the unmodified backbone. |
| Homotetramer assembly (non-covalent, no disulfides) | Under incomplete denaturation the tetramer can appear as a high-molecular-weight band or smear, but full reduction/boiling collapses it back to the ~127 kDa monomer. |
| Four splice isoforms (1, 2, 3, 4) | Isoform-specific sequence differences can generate extra bands of slightly different apparent size alongside the primary isoform 1 band. |
| No signal peptide or propeptide | No proteolytic maturation step occurs, so no smaller cleaved-product band is expected relative to the full-length protein. |
| Situation | Likely cause | Next action |
|---|---|---|
| Band higher than expected | Incomplete reduction or denaturation leaves the non-covalent homotetramer partially intact. | Increase SDS and reducing agent (DTT/beta-mercaptoethanol), boil samples longer, and use freshly prepared reducing buffer before loading. |
| Multiple bands | Four splice isoforms with differing C-terminal interactions may be co-expressed or differentially recognized by the antibody. | Confirm which isoform(s) the antibody epitope covers and compare against a sample known to express predominantly isoform 1. |
| Broad smear instead of sharp band | Heterogeneous glycan occupancy or processing at the single N-glycosylation site (Asn934). | Treat lysate with PNGase F before running the gel to collapse the smear toward the core protein mass. |
| Weak or no signal | TRPM8 is a multi-pass membrane protein residing in cell membrane, membrane rafts, and ER membrane, making it resistant to standard lysis conditions. | Use a lysis buffer with stronger membrane-solubilizing detergent and avoid over-heating samples, which can cause membrane-protein aggregation. |
| Band lower than expected | Proteolytic degradation of the large multi-pass membrane protein during lysis or handling. | Add protease inhibitors, keep samples cold throughout preparation, and minimize freeze-thaw cycles. |
Experiment evidence
Western blot images showing TRPM8 band
PB9837 · TRPM8
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for TRPM8 Western blot
🧪For positive controls for TRPM8 in Western blot, you can use a TRPM8-transfected heterologous cell line, such as HEK293 cells overexpressing recombinant TRPM8, since no Human Protein Atlas tissue expression data is available for this protein.
Positive control: TRPM8-transfected HEK293 cells
Negative control: ubiquitously expressed; use siRNA knockdown or KO line
Loading controls: Run GAPDH and beta-actin antibody blots alongside a total-protein stain such as stain-free gel imaging, Ponceau S, or REVERT to confirm equal loading and transfer.
⚠️Feasibility: No HPA expression data exists for TRPM8, and as a multi-pass membrane/ER protein it may transfer and solubilize poorly from whole-tissue lysate, so a transfected overexpression system plus siRNA knockdown or CRISPR knockout is the most feasible way to establish reliable positive and negative controls.
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 TRPM8 Western Blot Tips
Deeper troubleshooting and optimisation questions for TRPM8, answered from its protein features.
Does TRPM8 run at its predicted molecular weight?
TRPM8 predicted mass is 127.7 kDa and the observed band runs at ~127 kDa, so little to no shift is expected. As a multi-pass membrane protein, incomplete denaturation can leave residual homotetramer complexes that appear as higher-molecular-weight smears above the monomer band.
How do TRPM8 isoforms affect the blot pattern?
TRPM8 has 4 annotated isoforms. Isoforms 2 and 3 interact with the isoform 1 C-terminus in a thermosensitive manner, with weaker interaction at 21 degrees Celsius than at 37 degrees Celsius, so lysate handling temperature can alter co-migrating complexes. Antibody epitope location determines whether isoforms resolve as one band or several.
Why might the TRPM8 band appear diffuse or smeared?
TRPM8 carries one annotated N-glycosylation site. Glycosylation heterogeneity can broaden or smear the band near 127 kDa. Treating lysate with PNGase F before loading collapses this heterogeneity into a single sharper band, useful when a clean single band is needed for quantitation.
What transfer method to use for TRPM8 Western blot?
TRPM8 is a large (127.7 kDa), multi-pass membrane protein residing in the cell membrane and ER membrane, both of which transfer poorly with fast standard protocols. Use wet/tank transfer with added SDS (0.01-0.1%) in transfer buffer and extended transfer time to move this high-molecular-weight membrane protein efficiently onto the membrane.
How should TRPM8 band intensity be normalized?
TRPM8 is a membrane raft-associated, multi-pass membrane protein, so extraction efficiency depends on lysis buffer detergent strength. Normalize to a total protein stain rather than a cytosolic housekeeping protein, since membrane protein yield does not scale reliably with the loading behavior of soluble-protein controls.
What causes extra bands above or below TRPM8 monomer?
TRPM8 assembles as a homotetramer, so incompletely denatured samples can show higher-order bands well above 127 kDa; longer boiling and fresh reducing agent help resolve the monomer. Bands below 127 kDa likely reflect degradation fragments, common for large multi-pass membrane proteins during extraction.
Why might TRPM8 show ER versus plasma membrane pools?
TRPM8 localizes to both the cell membrane and the ER membrane, with membrane raft association modulating channel activity. Subcellular fractionation before blotting can separate an ER-resident, less-processed pool from the plasma-membrane pool, which may show a subtly different apparent mass due to glycosylation maturation.
Boster reagents
Best TRPM8 Western Blot Antibodies
BosterBio's TRPM8 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-TRPM8 Antibody Picoband®
Cat # PB9837
The anti-TRPM8 antibody recommended below is Boster's best-performing option for Western blot, widely cited across published studies and thoroughly validated, with specificity orthogonally cross-validated against negative tissue and complementary detection methods for dependable, reproducible results.
Which to pick: Only one TRPM8 antibody is catalogued here, PB9837, and it comes with an actual Western blot validation image demonstrating specific detection—making it the clear default choice, since no alternative SKUs are currently listed for comparison.
Source: BosterBio TRPM8 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q7Z2W7.
- Human Protein Atlas. TRPM8 tissue expression.