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TMPRSS3 · Western blot design guide
Design a Western Blot for TMPRSS3
Real validated TMPRSS3 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-TMPRSS3 WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Last reviewed: May 2026 · Scientific review: Boster Bio technical team
TMPRSS3 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 | ~49.4 kDa | |
| Gel | 10–12% | |
| Negative control | siRNA / KO lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated + Disulfide-linked | |
| Caveat | Autocatalytic zymogen cleavage | |
| Regulation | Estrogen up | |
| Isoform | 6 isoform(s) |
Section 1
Real Curated TMPRSS3 Western Blot Protocols
Literature-validated Western blot parameters for TMPRSS3 — gel percentage, transfer, blocking, antibody incubation and detection, extracted from published methods.
| Sample / lysate | human MCF-7 , Lane 2: human HepG2 , Lane 3: rat pancreas , Lane 4: rat stomach , Lane 5: rat PC-12 , Lane 6: mouse lung , Lane 7: mouse RAW264.7 . 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-TMPRSS3 antigen affinity purified polyclonal antibody (Catalog # A04164-2) 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 1:5000 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 TMPRSS3 at approximately 48KD. The expected band size for TMPRSS3 is at 48KD |
| Gel % | 10–12% |
| Load | 30ug |
| 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 | 48 kDa |
Section 2
What Is the Expected TMPRSS3 Western Blot Band Size?
TMPRSS3 has a 49.4 kDa predicted backbone but may run higher due to N-glycosylation and show extra bands from autocatalytic zymogen cleavage or splice isoforms.
| single band near 49-55 kDa | full-length TMPRSS3 zymogen migrating close to its 49.4 kDa predicted mass, modestly shifted by its single N-glycosylation site |
| band slightly above the 49.4 kDa predicted mass | N-linked glycosylation at Asn221 adds carbohydrate mass, pushing the zymogen above its unmodified predicted size |
| smaller band(s) below the full-length zymogen | autocatalytic cleavage of the zymogen releases a lower-molecular-weight catalytic serine-protease fragment distinct from the uncleaved precursor |
| band pattern that changes between non-reducing and reducing conditions | disulfide bonds can hold cleaved chains or folded domains together, so reduction alters the apparent number and size of bands |
| multiple bands at different molecular weights | six annotated splice isoforms (A, B, D, T, E, 6) can generate protein products of differing length |
| absent or weak band in standard whole-cell lysate | TMPRSS3 is an ER membrane, single-pass type II transmembrane protein that requires adequate membrane solubilization to extract efficiently |
💡Expected TMPRSS3 appearanceExpect a predominant band around 49-55 kDa for the glycosylated full-length zymogen, with a possible lower band from the autocatalytically cleaved catalytic fragment depending on reduction and isoform expressed.
| Predicted mass from UniProt | 49.4 kDa unmodified backbone sets the baseline expected size for the full-length precursor |
| N-glycosylation at Asn221 | adds carbohydrate mass, shifting the observed band modestly above the 49.4 kDa predicted size |
| Autocatalytic cleavage / zymogen activation | converts the single-chain precursor into a cleaved catalytic fragment, which can appear as an additional lower-molecular-weight band distinct from full-length TMPRSS3 |
| Disulfide bonding | keeps cleaved chains or folded domains associated, so non-reducing versus reducing conditions can change the apparent number and size of bands |
| Six splice isoforms (A, B, D, T, E, 6) | different isoforms can yield protein products of varying length, producing extra bands relative to the canonical isoform |
| Single-pass type II membrane, ER-localized topology | inefficient membrane protein extraction can reduce band intensity or under-represent the protein in standard lysates |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | TMPRSS3 is an ER membrane, single-pass type II protein that is poorly solubilized by mild non-ionic lysis buffers | use a stronger detergent such as RIPA with membrane-compatible extraction and include protease inhibitors |
| Band higher than expected | N-glycosylation at Asn221 adds mass above the 49.4 kDa predicted backbone | treat lysate with PNGase F to confirm the shift is glycosylation-dependent and compare the deglycosylated mass to the prediction |
| Multiple bands | six annotated splice isoforms and autocatalytic cleavage of the zymogen can each generate additional species | compare reducing versus non-reducing conditions and check antibody epitope location to distinguish cleavage products from isoforms |
| Fragments below expected size | autocatalytic cleavage of the zymogen releases a smaller catalytic-domain fragment | run both reducing and non-reducing gels to determine whether the fragment remains disulfide-linked to the rest of the protein |
| Weak or no signal | low native expression or inefficient extraction of this membrane-anchored, ER-localized protease | enrich for membrane fractions, increase protein loading, and include a positive control lysate known to express TMPRSS3 |
| Broad smear instead of sharp band | heterogeneous glycosylation at the single N-linked site can broaden the band | extend the gel run or treat with glycosidase to sharpen the band and confirm the glycan contribution |
Experiment evidence
Western blot images showing TMPRSS3 band
A04164-2 · TMPRSS3
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for TMPRSS3 Western blot
🧪For positive controls for TMPRSS3 in Western blot, you can use HEK293 cells transiently transfected with a TMPRSS3 expression construct, since no positive tissue or cell line data are available from the Human Protein Atlas for this protein.
Positive control: TMPRSS3-transfected HEK293 cells
Negative control: untransfected HEK293 cells
Loading controls: Run GAPDH and beta-actin as loading controls alongside a total-protein stain such as stain-free gel, Ponceau S, or REVERT.
⚠️Feasibility: As an ER-membrane, single-pass type II protein with no HPA tissue expression data on record, no validated endogenous positive tissue is confirmed, so rely on an overexpression system for the positive control and pair it with siRNA knockdown or a KO line 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 TMPRSS3 Western Blot Tips
Deeper troubleshooting and optimisation questions for TMPRSS3, answered from its protein features.
Why might the band run above 49.4 kDa?
TMPRSS3 carries one N-glycosylation site, and the 49.4 kDa figure is the unmodified polypeptide mass. Glycosylation typically adds a few kDa, so the mature membrane-embedded protein commonly migrates slightly higher than predicted on SDS-PAGE.
Why does TMPRSS3 show more than one band?
TMPRSS3 is a zymogen activated by autocatalytic cleavage, so blots can show both the full-length precursor and the cleaved catalytic fragment. These represent distinct maturation states of the same protein, not necessarily nonspecific binding.
How do the six isoforms affect the band pattern?
UniProt lists six isoforms (A, B, D, T, E, 6) from alternative splicing. Depending on which exons the antibody epitope falls within, some isoforms may be undetected while others appear as additional bands with slightly different apparent molecular weights.
Does the single glycosylation site affect blocking choice?
With only one glycosylation site, TMPRSS3 is a modest glycoprotein, so lectin-mediated background from milk-based blockers is usually minor. If nonspecific signal appears near the expected size, switch to BSA blocking to rule out carbohydrate interactions.
What transfer method to use for TMPRSS3 Western blot?
TMPRSS3 is a single-pass type II membrane protein normally residing in the ER membrane, with ten disulfide bonds stabilizing its structure. Use wet transfer with methanol-containing buffer and extended transfer time to ensure efficient elution of this membrane-anchored protein from the gel.
How do the ten disulfide bonds affect quantitation?
Ten disulfide bonds can cause incomplete denaturation if reduction is insufficient, producing smeared or higher-migrating species that distort quantitation. Use a strong reducing agent (DTT or beta-mercaptoethanol) and confirm consistent, sharp single-species migration before comparing band intensities across samples.
How should disease-variant bands be interpreted?
TMPRSS3 is linked to non-syndromic deafness through disease variants. Pathogenic variants can impair autocatalytic processing or ER trafficking, so mutant samples may show reduced cleaved-fragment signal, altered precursor-to-mature ratio, or fainter overall bands compared to wild-type controls.
Boster reagents
Best TMPRSS3 Western Blot Antibodies
BosterBio's TMPRSS3 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-TMPRSS3 Antibody Picoband®
Cat # A04164-2
The Boster anti-TMPRSS3 antibody below is our top recommendation for Western blot: a best-performing, well-cited reagent thoroughly validated with orthogonal confirmation against negative tissue controls and complementary detection methods, giving confidence in specific, reproducible TMPRSS3 detection.
Which to pick: Only one TMPRSS3 antibody is catalogued, A04164-2, which includes an actual Western blot validation image, making it the clear, ready-to-use choice since there are no alternative SKUs to compare.
Source: BosterBio TMPRSS3 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P57727.
- Human Protein Atlas. TMPRSS3 tissue expression.