Anti-TMPRSS-5 Rabbit Polyclonal Antibody

Frequently Asked Questions

What is the expected molecular weight for TMPRSS-5 on Western blot?

TMPRSS-5 migrates at approximately 50-52 kDa on reduced SDS-PAGE, corresponding to the full-length 457-amino acid transmembrane protease. As a type II transmembrane protein with post-translational modifications including potential glycosylation, you may observe slight upward shifts or diffuse bands depending on cell type and expression levels. Some lysates show a minor band near 30 kDa representing the shed catalytic domain following autoproteolysis, though this is variable. Start with 1:1000 dilution in 5% non-fat milk and ensure adequate blocking to minimize background from the extracellular region epitopes.

Which human tissues or cell lines express TMPRSS-5 at detectable levels?

TMPRSS-5 expression is highest in inner ear tissues, particularly cochlear epithelium, consistent with its predicted role in auditory function. In standard cell line panels, expression is generally low to moderate. HEK293 cells transfected with TMPRSS-5 serve as a positive control; endogenous levels in common cancer lines are often below Western blot detection limits without enrichment. For initial validation, consider using inner ear cDNA or lysates from cells known to express related TMPRSS family members. Immunohistochemistry on formalin-fixed cochlear tissue sections may yield more robust signals than lysate-based methods.

Does this TMPRSS-5 antibody cross-react with other TMPRSS family proteases?

The antibody is raised against a region spanning residues corresponding to the TMPRSS-5 extracellular and catalytic domains. While validated specifically for human TMPRSS-5, cross-reactivity with closely related family members such as TMPRSS-2, TMPRSS-4, or matriptase has not been systematically ruled out. If your sample contains multiple TMPRSS proteases, run parallel blots with lysates from cells individually overexpressing each isoform to confirm specificity. Predicted cross-reactivity with monkey orthologs reflects high sequence homology in the immunogen region, but experimental validation in primate samples remains advisable.

What is the recommended starting dilution for TMPRSS-5 Western blot experiments?

We recommend starting at 1:1000 dilution for Western blot with overnight incubation at 4°C. This has been validated with whole-cell lysates containing 20-40 µg total protein per lane. Depending on endogenous expression levels in your system, you may need to optimize between 1:500 and 1:2000. TMPRSS-5 is a low-to-moderate abundance membrane protein, so use RIPA or detergent-containing buffers to ensure efficient solubilization. Include a recombinant TMPRSS-5 standard or transfected lysate as a positive control, especially if working with untested cell types where expression may be undetectable.

Can I use this antibody for immunofluorescence or immunohistochemistry on TMPRSS-5?

This rabbit polyclonal is validated for Western blot and suitable for immunohistochemistry on formalin-fixed paraffin-embedded sections. For IHC, begin with 1:100 to 1:200 dilution and include antigen retrieval in citrate buffer, pH 6.0, to unmask epitopes. Immunofluorescence on fixed cells is feasible but requires empirical titration starting around 1:50 to 1:100. Because TMPRSS-5 is a transmembrane protein, permeabilization with 0.1-0.3% Triton X-100 is essential for intracellular epitope access. Background can be higher in IF than Western blot; always include secondary-only and non-expressing cell controls.

How should I prepare lysates to preserve TMPRSS-5 catalytic activity and epitope integrity?

TMPRSS-5 is a serine protease capable of autoproteolysis and substrate cleavage, which can complicate detection. Lyse cells rapidly on ice with complete protease inhibitor cocktails including serine protease inhibitors such as PMSF or aprotinin. RIPA buffer with 1% NP-40 and 0.1% SDS efficiently solubilizes membrane fractions. Avoid repeated freeze-thaw cycles, which promote aggregation and proteolytic degradation. If studying the active protease, separate experiments under non-reducing conditions may reveal zymogen versus mature forms. For routine detection of total TMPRSS-5, reducing conditions with beta-mercaptoethanol or DTT and boiling for five minutes are appropriate.

What are appropriate positive and negative controls for TMPRSS-5 antibody validation?

The ideal positive control is a lysate from HEK293 or HeLa cells transiently transfected with full-length human TMPRSS-5 expression plasmid, which should yield a clear 50-52 kDa band. Recombinant TMPRSS-5 protein, if available, provides a second standard. Negative controls include untransfected parental cells and lysates from tissues with negligible TMPRSS expression, such as erythrocytes. siRNA or CRISPR knockdown of TMPRSS-5 in an expressing line offers the strongest specificity control. For IHC, compare staining in TMPRSS-5-positive cochlear tissue versus liver or skeletal muscle, where expression is predicted to be absent.

How should I store the TMPRSS-5 antibody and what is the typical shelf life?

Store the antibody at -20°C in single-use aliquots to avoid freeze-thaw cycles, which denature immunoglobulins and reduce titer. The 100 µg pack size is sufficient for approximately 100 Western blots at 1:1000 dilution. After thawing an aliquot, keep at 4°C for up to one month if supplemented with 0.02% sodium azide as preservative. Do not store diluted antibody in blocking buffer for more than 24 hours. Typical shelf life is 12 months from manufacture when stored properly. If signal weakens over time, verify by running a fresh aliquot alongside aged stock against a consistent positive control lysate.