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- Table of Contents
TMPRSS2 / Transmembrane protease serine 2 · Western blot design guide
Design a Western Blot for TMPRSS2
Real validated TMPRSS2 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-TMPRSS2 WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Evidence assembled September 2026 · For research use; verify linked source records and product datasheet before use
TMPRSS2 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 | ~53.9 kDa | |
| Observed band | ~54 kDa | |
| Gel | 5–20% (catalog M00666) | |
| Positive control | Kidney (IHC candidate; verify WB) +4 more | |
| Negative control | Adipose tissue (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated | |
| Caveat | Autocleavage controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated TMPRSS2 Western Blot Protocols
The M00666 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human LNCAP, human COLO-320, human RT4 (catalog M00666) |
| Gel % | 5–20% (catalog M00666) |
| Load | 30 ug; reducing conditions (catalog M00666) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog M00666) |
| Membrane | nitrocellulose membrane (catalog M00666) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog M00666) |
| Primary antibody | M00666 · 1:500 (catalog M00666) |
| Primary incubation | overnight at 4°C (catalog M00666) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog M00666) |
| Secondary incubation | 1.5 hour at RT (catalog M00666) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog M00666) |
| Detection | ECL (catalog M00666) |
Section 2
What Is the Expected TMPRSS2 Western Blot Band Size?
TMPRSS2 is predicted at 53.9 kDa and observed at ~54 kDa; the cause of their small difference is not established.
| Band at ~54 kDa | Empirical TMPRSS2 band, consistent with the 53.9 kDa predicted mass |
| Additional band above ~54 kDa | Could reflect N-linked glycosylation at Asn213 or Asn249; identity requires confirmation |
| Additional bands at different positions | Could reflect isoforms 1 and 2, but distinct migration is not established |
| Bands below ~54 kDa | Could reflect autocatalytic cleavage; fragment sizes are not supplied |
| Weak band in soluble lysate | Membrane-localized TMPRSS2 may be poorly extracted |
💡Expected TMPRSS2 appearanceUniProt predicts 53.9 kDa, and antibody QC detects a band at ~54 kDa in reducing whole-cell and tissue lysates; confirm identity with appropriate positive and antibody controls.
| UniProt predicted mass | 53.9 kDa provides the sequence-based reference; the empirical band is ~54 kDa |
| N-linked glycosylation at Asn213 | May affect apparent size, but no visible shift is established |
| N-linked glycosylation at Asn249 | May affect apparent size, but no visible shift is established |
| Splice isoforms 1 and 2 | May differ in size; their masses and migration are not supplied |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Membrane-localized protein may be poorly extracted | Check membrane extraction and run a positive lysate |
| Band higher than expected | N-linked glycosylation is possible, but its migration effect is unproven | Compare with deglycosylated sample and confirm antibody specificity |
| Band lower than expected | Autocatalytic cleavage is possible; fragment identity is unproven | Compare antibodies recognizing different regions and use protease inhibitors |
| Broad smear instead of sharp band | Heterogeneous glycosylation is possible at the two N-linked sites | Compare before and after deglycosylation and check sample quality |
| Multiple bands | Isoforms or cleavage products are possible, but their migration is unknown | Use region-specific antibodies and a positive control to identify bands |
| Weak or no signal | Membrane protein recovery may be low | Check extraction, loading, and a positive lysate |
| Fragments below expected size | Autocatalytic cleavage or sample proteolysis may contribute | Use protease inhibitors and compare antibodies recognizing different regions |
Experiment evidence
Western blot images showing TMPRSS2 band
M00666 · TMPRSS2
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for TMPRSS2 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for TMPRSS2 in Western blot, you can use kidney tissue, which has high HPA expression.
Positive control: Kidney (IHC candidate; verify WB)
Negative control: Adipose tissue (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: Because TMPRSS2 is membrane-bound and can be extracellular, sample preparation may affect signal strength.
HPA tissue expression evidence for TMPRSS2
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.
Higher expression tissues · candidate positive controls from IHC
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Kidney | proximal tubules (microvilli) | High | Protein (IHC) | HPA → |
| Rectum | enterocytes - Microvilli | High | Protein (IHC) | HPA → |
| Colon | enterocytes - Microvilli | Medium | Protein (IHC) | HPA → |
| Epididymis | glandular cells | Medium | Protein (IHC) | HPA → |
| Pancreas | exocrine glandular cells | Medium | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | adipocytes | Not detected | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | adipocytes | Not detected | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced TMPRSS2 Western Blot Tips
Deeper troubleshooting and optimisation questions for TMPRSS2, answered from its protein features.
How should TMPRSS2 band migration be interpreted?
Band shift · Use the separately labelled calculated mass and catalog-observed evidence above. A sequence annotation does not establish an observed migration shift. Verify target identity with orthogonal controls.
Could TMPRSS2 isoforms produce different bands?
Isoforms · UniProt lists isoforms 1 and 2. In isoform 2, the initial methionine is replaced by MPPAPPGGESGCEERGAAGHIEHSRYLSLLDAVDNSKM. Check which isoform the antibody recognizes before assigning bands; the supplied features do not establish where either isoform migrates.
Which glycosylation sites matter when interpreting TMPRSS2 bands?
PTM · UniProt lists N-linked glycosylation at Asn213 and Asn249, using canonical sequence coordinates. If comparing site labels from an antibody or paper, check its numbering convention before assigning a band. The annotations do not specify the size of any shift.
Does this guide establish induction of TMPRSS2?
Induction · No general induction response is established by this guide. A pathway or gene-set association is not evidence of induction in a particular specimen. Verify the relevant treatment and control in a target-specific experiment.
How should transfer be checked for TMPRSS2?
Transfer · Standard workflow guidance: verify transfer efficiency for the intended target size before interpreting a weak signal. Use total-protein assessment and optimize transfer for the membrane, gel and apparatus; the labelled catalog values take precedence.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the M00666 datasheet where specified. Otherwise compare 5% milk or 5% BSA in TBST; for a phospho-specific assay start with BSA. Optimize background and specific signal with matched controls.
How should TMPRSS2 bands be quantified across samples?
Quantitation · Define the approximately 54 kDa band consistently and use the same sample preparation and reducing conditions across lanes. TMPRSS2 has two isoforms and an autocatalytic cleavage annotation, so quantify additional bands separately until their identities are established.
Why is the TMPRSS2 band near 54 kDa?
Interpretation · The supplied apparent band is approximately 54 kDa, close to the predicted 53.9 kDa. TMPRSS2 has two annotated N-linked glycosylation sites, but their presence alone does not establish a visible shift or explain a mass difference.
TMPRSS2 is annotated as a zymogen with autocatalytic cleavage. A smaller band could warrant investigation as a processed form, but the supplied features give no cleavage position or fragment mass. Check antibody epitope coverage before assigning its identity.
UniProt lists nine disulfide bonds. Keep reducing conditions consistent across samples, and compare reducing and nonreducing preparations if band patterns differ. The supplied features do not predict a specific mobility change.
TMPRSS2 is annotated as a zymogen with autocatalytic cleavage. A smaller band could warrant investigation as a processed form, but the supplied features give no cleavage position or fragment mass. Check antibody epitope coverage before assigning its identity.
UniProt lists nine disulfide bonds. Keep reducing conditions consistent across samples, and compare reducing and nonreducing preparations if band patterns differ. The supplied features do not predict a specific mobility change.
Boster reagents
TMPRSS2 Western Blot Antibodies
Catalog antibodies with Western blot application and product-specific WB images. Evaluate suitability with the reported sample, controls and experimental conditions.
Real WB data
Anti-TMPRSS2 Rabbit Monoclonal Antibody
Cat # M00666
The catalog reports one anti-TMPRSS2 rabbit monoclonal antibody, M00666, with reported human, mouse, and rat reactivity. Its WB image shows an approximately 54 kDa band in four human cell lysates and rat kidney lysate; the supplied caption does not show mouse testing.
Which to pick: M00666 is the only listed option. Its WB image documents human LNCAP, COLO-320, RT4, and Caco-2 lysates plus rat kidney lysate, using 30 µg per lane under reducing conditions and a 1:500 primary dilution. Mouse reactivity is listed without a mouse WB example.
Source: BosterBio TMPRSS2 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry O15393.
- Human Protein Atlas. TMPRSS2 tissue expression.
- PMC9961850 — target-verified WB comparison
- PMC5215063 — target-verified WB comparison
- PMC5609883 — target-verified WB comparison