Anti-Matriptase-2 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight band should I expect for Matriptase-2 on Western blot?

Full-length Matriptase-2 (TMPRSS6) migrates at approximately 90-95 kDa on reducing SDS-PAGE, consistent with the 811-residue type II transmembrane protease structure. You may also detect proteolytic fragments around 60-70 kDa, representing the shed serine protease domain after autocatalytic cleavage. Band intensity and fragment pattern depend on cell type and activation state. Hepatocyte lysates typically show both forms. If you observe only lower molecular weight species, consider whether your sample contains active protease that has undergone maturation. Always include a positive control lysate from liver or hepatocyte lines known to express TMPRSS6.

Does this Matriptase-2 antibody cross-react with mouse or monkey samples?

This polyclonal antibody is validated for human Matriptase-2 and predicted to cross-react with mouse and monkey based on sequence homology in the immunogen region. Human and mouse TMPRSS6 share approximately 75 percent amino acid identity overall, with higher conservation in the catalytic domain. For mouse samples, start with the recommended 1:1000 dilution and optimize empirically. Monkey reactivity is predicted but not experimentally validated. If working with non-human samples, include a known positive control and be prepared to adjust dilution or blocking conditions. We recommend pilot experiments to confirm signal specificity before committing to large sample sets.

What is the recommended starting dilution for Western blot with this antibody?

Start at 1:1000 dilution in blocking buffer for Western blot of human samples. This polyclonal was raised in rabbit and typically provides robust signal at this concentration when detecting endogenous Matriptase-2 in hepatocyte or liver lysates. If working with overexpression systems or samples with high TMPRSS6 abundance, you may achieve adequate signal at 1:2000 or even 1:5000. Conversely, samples with low expression may require 1:500. Incubate overnight at 4°C or two hours at room temperature. Use standard HRP-conjugated anti-rabbit secondary at 1:5000 to 1:10000. Titrate against your specific lysate to balance signal and background.

Which tissues or cell lines serve as good positive controls for Matriptase-2?

Liver tissue and primary hepatocytes are the gold standard positive controls, as TMPRSS6 is predominantly expressed in the liver where it regulates hepcidin production. Human hepatocyte cell lines such as HepG2 or Huh7 also express detectable Matriptase-2, though levels may be lower than primary cells. Duodenal enterocytes show modest expression. Avoid using fibroblasts, lymphocytes, or most cancer cell lines unless they are known to aberrantly express TMPRSS6. For negative controls, consider tissues with negligible expression such as brain or skeletal muscle. If developing an iron metabolism model, compare wild-type versus TMPRSS6-knockdown cells to confirm antibody specificity.

Can this antibody detect the difference between active and inactive Matriptase-2?

This polyclonal recognizes epitopes in the full-length protein and likely binds both zymogen and cleaved forms, so it will not selectively detect only active Matriptase-2. The enzyme undergoes autocatalytic activation that releases the serine protease domain from the membrane-bound stem region. On Western blot you may observe multiple bands corresponding to precursor and processed forms, but distinguishing zymogen from active protease requires functional assays or antibodies specific to neo-epitopes generated during activation. If your research question centers on activation state, consider complementing immunoblotting with activity assays measuring hemojuvelin cleavage or hepcidin reporter readouts in cultured hepatocytes.

What sample preparation considerations are important for detecting Matriptase-2?

Because Matriptase-2 is a type II transmembrane protease, use lysis buffers containing non-ionic detergent such as 1 percent Triton X-100 or NP-40 to solubilize membrane proteins effectively. RIPA buffer works well. Include protease inhibitors to prevent autodegradation, particularly if studying the zymogen form. For tissue samples, snap-freeze in liquid nitrogen immediately after harvest to preserve protein integrity. Liver homogenates should be clarified by centrifugation at 10,000 to 15,000 g for ten minutes at 4°C. Load 20 to 50 micrograms total protein per lane for endogenous detection. Avoid repeated freeze-thaw cycles, which can promote aggregation or cleavage.

How should I store this Matriptase-2 antibody and what is the expected shelf life?

Store the antibody at minus 20°C in small aliquots to avoid repeated freeze-thaw cycles, which can reduce titre and increase aggregation. Glycerol or BSA in the formulation typically stabilizes polyclonals during storage. Once thawed, an aliquot remains stable at 4°C for up to one month; for longer bench use, add 0.02 to 0.05 percent sodium azide as preservative. Do not store diluted antibody for more than a few days. Under these conditions, rabbit polyclonals generally retain activity for two to three years from manufacture date. If you observe loss of signal or increased background over time, prepare a fresh aliquot and re-optimize dilution.

Does Matriptase-2 expression vary with iron status in hepatocyte cultures?

TMPRSS6 expression itself is relatively stable, but the functional significance of Matriptase-2 becomes more apparent under iron-deficient conditions when the hepcidin-suppressive pathway is engaged. In cultured hepatocytes, iron depletion or treatment with iron chelators does not dramatically upregulate TMPRSS6 mRNA or protein in most studies, unlike the robust induction seen with transferrin receptor 1. Instead, the protease activity toward hemojuvelin becomes physiologically critical. If modeling iron homeostasis, compare hepcidin output and hemojuvelin cleavage rather than total Matriptase-2 protein. For Western blot experiments, baseline expression in standard culture medium should be readily detectable without iron manipulation.