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- Table of Contents
TRPC5 · Western blot design guide
Design a Western Blot for TRPC5
Real validated TRPC5 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-TRPC5 WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Last reviewed: May 2026 · Scientific review: Boster Bio technical team
TRPC5 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 | ~111.4 kDa | |
| Observed band | ~111 kDa | |
| Gel | 8–10% | |
| Negative control | siRNA / KO lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated | |
| Caveat | Homotetramer oligomerization | |
| Regulation | LPS-suppressed | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated TRPC5 Western Blot Protocols
Literature-validated Western blot parameters for TRPC5 — gel percentage, transfer, blocking, antibody incubation and detection, extracted from published methods.
| Sample / lysate | human placenta , Lane 2: human U-87MG , Lane 3: human K562 , Lane 4: human A549 , Lane 5: human Caco-2 . 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-TRPC5 antigen affinity purified polyclonal antibody (Catalog # A02701) 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 TRPC5 at approximately 111KD. The expected band size for TRPC5 is at 111KD |
| Gel % | 8–10% |
| Load | 50ug |
| Transfer | nitrocellulose membrane, 150 mA, 50–90 min |
| Membrane | nitrocellulose |
| Blocking | 5% non-fat milk / TBS, 1.5 h RT |
| 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 | 111 kDa |
Section 2
What Is the Expected TRPC5 Western Blot Band Size?
TRPC5 has a predicted mass of 111.4 kDa and runs at the same ~111 kDa on reducing SDS-PAGE, with only a minor shift from its single N461 glycosylation site.
| single sharp band at ~111 kDa | matches the calculated mass of the 973-residue TRPC5 polypeptide with minimal net shift from modification |
| band running slightly above the unmodified 111.4 kDa backbone or showing a faint upper shoulder | reflects the single N-linked glycosylation site at Asn461 adding modest mass |
| higher-molecular-weight species (multiples of ~111 kDa) under non-reducing or incompletely denaturing conditions | reflects the native homotetrameric channel assembly (ANK-repeat mediated) not fully dissociated by SDS/heat |
| no extra bands attributable to alternate transcripts | only one annotated isoform exists for TRPC5, so multi-band patterns are unlikely to come from splicing |
| very faint or absent signal in some whole-cell lysates despite loading | TRPC5 is a multi-pass cell-membrane channel protein that can be under-solubilized by mild lysis conditions |
💡Expected TRPC5 appearanceExpect a single band at approximately 111 kDa on reducing SDS-PAGE, closely matching the calculated 111.4 kDa mass of the 973-residue polypeptide with only a minor shift from its single N-glycosylation site.
| Predicted mass (111.4 kDa, 973 aa) | sets the baseline expected migration position on SDS-PAGE |
| N-linked glycosylation at Asn461 | adds modest mass, can nudge the band slightly above the unmodified backbone or add slight heterogeneity |
| Homotetrameric channel assembly (non-covalent, via ANK repeats) | can leave higher-order oligomeric species on the gel if denaturation is incomplete, though full reducing/denaturing treatment collapses it to the ~111 kDa monomer |
| Intrachain disulfide bond (Cys553-Cys558) | stabilizes a local loop within the monomer rather than linking separate subunits, so it does not itself generate a dimer-sized band |
| Recombinant/truncated antigen standards | smaller recombinant fragments used in some antibody QC lanes run well below the native full-length size and should not be used to judge expected native band position |
| Situation | Likely cause | Next action |
|---|---|---|
| Band higher than expected | incomplete denaturation of the non-covalent homotetrameric channel assembly | boil samples longer in fresh reducing SDS sample buffer with DTT or beta-mercaptoethanol before loading |
| Band lower than expected | comparison against a truncated or non-native recombinant standard rather than full-length native TRPC5 | confirm the antibody immunogen region and benchmark lysate bands against a full-length native positive control, not a recombinant fragment |
| Broad smear instead of sharp band | heterogeneous glycosylation at the single N461 site or aggregation of this multi-pass membrane protein | run a PNGase F deglycosylation control and optimize lysis/solubilization to limit aggregation |
| Weak or no signal | poor solubilization of a multi-pass membrane protein by standard lysis buffers | use a detergent-based lysis buffer suited to membrane proteins and increase total protein loaded |
| Multiple bands | partial proteolytic degradation of the large 973-residue membrane protein during extraction | add fresh protease inhibitors and keep samples cold throughout lysis and processing |
Experiment evidence
Western blot images showing TRPC5 band
A02701 · TRPC5
PB9271 · TRPC5
PB9271 · TRPC5
PB9271 · TRPC5
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for TRPC5 Western blot
🧪For positive controls for TRPC5 in Western blot, you can use HEK293 cells transiently transfected with a TRPC5 expression construct, since no Human Protein Atlas expression data are available for this protein.
Positive control: TRPC5-transfected HEK293 cells
Negative control: ubiquitously expressed; use siRNA knockdown or KO line
Loading controls: Also probe for GAPDH and β-actin as loading controls alongside a total-protein stain (e.g., stain-free gel, Ponceau S, or REVERT).
⚠️Feasibility: As a multi-pass membrane ion channel with no Human Protein Atlas tissue data, TRPC5 lacks a well-validated endogenous positive tissue, so overexpression systems and siRNA/KO controls are needed to confirm antibody 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 TRPC5 Western Blot Tips
Deeper troubleshooting and optimisation questions for TRPC5, answered from its protein features.
Why does TRPC5 run at its predicted 111 kDa size?
TRPC5's predicted mass of 111.4 kDa matches the observed ~111 kDa band, indicating little mobility shift from post-translational modification. With only one glycosylation site and no other modified residues annotated, the protein migrates close to its calculated molecular weight on standard SDS-PAGE.
Does TRPC5 have isoforms that alter band pattern?
UniProt lists only a single TRPC5 isoform, so a lone ~111 kDa band is expected. Extra bands should not be attributed to alternative splicing; instead investigate degradation, incomplete denaturation, or cross-reactivity with related TRPC subunits.
Does N-glycosylation affect the TRPC5 band position?
TRPC5 has one predicted N-glycosylation site, which can cause a small upward mobility shift depending on glycosylation state. No phosphorylation or other modified residues are annotated, so aside from this glycosylation effect, migration should closely track the theoretical mass.
How should blocking be optimized for TRPC5 detection?
Because TRPC5 is a glycoprotein, milk-based blockers (which contain endogenous glycoproteins) can raise background via lectin-like interactions with glycosylated targets or antibodies. Switching to a BSA-based blocking buffer typically reduces nonspecific signal around the 111 kDa region.
What transfer method to use for TRPC5 Western blot?
TRPC5 is a large (111 kDa), multi-pass membrane protein, which can transfer inefficiently. Wet/tank transfer with reduced methanol (~10%) and extended transfer time is recommended over rapid semi-dry methods to ensure complete elution of this hydrophobic protein from the gel.
Why must samples be fully reduced for TRPC5 quantitation?
TRPC5 assembles into homotetramers, or heterotetramers with TRPC1/TRPC4, and contains one disulfide bond. Incomplete reduction or denaturation can leave oligomeric complexes intact, causing signal to run higher than 111 kDa and skewing quantitation of the true monomer band.
What explains bands above the 111 kDa monomer?
Higher molecular weight bands can reflect incompletely dissociated TRPC5 homotetramers, heterotetramers with TRPC1/TRPC4, or disulfide-linked dimers if reducing agent is insufficient. Re-boiling samples in fresh reducing sample buffer usually collapses these species back to the monomeric 111 kDa band.
Boster reagents
Best TRPC5 Western Blot Antibodies
BosterBio's TRPC5 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-TRPC5 Antibody Picoband®
Cat # A02701
Real WB data
Anti-TRPC5 Antibody Picoband®
Cat # PB9271
These TRPC5 antibodies are top-performing Boster reagents, extensively cited and rigorously validated by Western blot, including orthogonal confirmation against recombinant protein controls and predicted molecular weight matching, giving researchers confident, reproducible detection across sample types.
Which to pick: Both A02701 and PB9271 include real Western blot validation images. Choose PB9271 for recombinant-protein-confirmed specificity with matching predicted/observed 50KD band; choose A02701 for validated detection in electrophoresed sample lysates using standard SDS-PAGE conditions.
Source: BosterBio TRPC5 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q9UL62.
- Human Protein Atlas. TRPC5 tissue expression.