Anti-Kallikrein-12 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight band should I expect for Kallikrein-12 on Western blot?

The mature secreted form of human KLK12 runs at approximately 28-30 kDa on reducing SDS-PAGE, corresponding to the processed 248-amino-acid catalytic chain after signal peptide and pro-peptide removal. You may also observe a band around 32-35 kDa representing the pro-enzyme form, particularly in cell lysates or conditioned media from overexpression systems. The appearance of pro- versus mature forms depends on your sample source and whether endogenous or exogenous convertases are present. Glycosylation is not a major contributor to KLK12 mass, so band shifts are typically limited. Always include a positive control lysate from KLK12-expressing tissue such as prostate or salivary gland.

What Western blot dilution should I start with for Kallikrein-12 antibody?

We recommend starting at 1:1000 dilution in blocking buffer (5 percent non-fat milk or BSA in TBST) for overnight incubation at 4°C. This polyclonal was validated at that dilution against human recombinant KLK12 and prostate lysate. If your target tissue or cell line expresses KLK12 at low levels, you may need to increase antibody concentration to 1:500 or load more total protein per lane. Conversely, if you see high background, try 1:2000. Secreted kallikreins are often low-abundance, so enriching conditioned media by ultrafiltration or using 30-50 µg total lysate can improve signal without pushing antibody concentration too high.

Does this Kallikrein-12 antibody cross-react with other kallikrein family members?

The tissue kallikrein family comprises 15 closely related serine proteases with significant sequence homology, so cross-reactivity is a valid concern. This polyclonal was raised against a recombinant fragment spanning a substantial portion of KLK12, and our validation blots show a single major band at the expected molecular weight in KLK12-positive lysates. However, we have not systematically tested all 14 other KLK family members. If you work with samples co-expressing KLK4, KLK5, KLK7, or KLK14, consider running KLK12 knockout or knockdown lysates as negative controls. Preabsorption with recombinant KLK12 can also confirm specificity if you observe unexpected bands.

Is this antibody suitable for detecting Kallikrein-12 in monkey or dog samples?

KLK12 orthologs in cynomolgus monkey and dog share approximately 85-90 percent amino acid identity with human KLK12, and we predict cross-reactivity based on epitope conservation within the immunogen region. However, validation in these species has not been performed in-house. If you plan to work with non-human primate or canine tissues, we recommend testing a dilution series starting at 1:500 alongside a positive human control. Rodent KLK12 orthologs diverge more substantially, and this antibody is not expected to recognize mouse or rat. Pan-mammalian reactivity is not guaranteed; empirical titration against your lysate is essential before committing to large experiments.

What positive control tissue or cell line should I use for Kallikrein-12 Western blots?

Human prostate tissue or prostate-derived cell lines such as LNCaP are reliable positive controls; KLK12 mRNA and protein are well documented in prostatic epithelium. Salivary gland tissue also expresses KLK12 at detectable levels. If working with conditioned media, LNCaP or PC-3 cells cultured in serum-free medium for 24-48 hours will secrete KLK12 into the supernatant, which you can concentrate by ultrafiltration. Stomach, colon, and lung tissues show lower but measurable expression. For a negative control, HEK293T or HeLa lysates typically have minimal endogenous KLK12. Include recombinant human KLK12 as a defined reference if available.

Can I use this antibody for immunohistochemistry on formalin-fixed paraffin-embedded sections?

This rabbit polyclonal has been validated for IHC applications, consistent with our broader antibody portfolio. For FFPE sections of human prostate, salivary gland, or gastrointestinal tissue, standard heat-induced epitope retrieval in citrate buffer (pH 6.0) or Tris-EDTA (pH 9.0) is recommended. Start with a 1:100 to 1:200 dilution and optimize based on signal intensity and background. Because KLK12 is a secreted protease, you should observe staining in the extracellular space and within secretory epithelial cells. Counterstain with hematoxylin, and always include a tissue known to lack KLK12 expression as a specificity control. Frozen sections generally give cleaner signal and require less optimization.

How should I store this Kallikrein-12 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. The formulation buffer typically contains glycerol and a preservative to maintain stability. Under these conditions, polyclonal antibodies retain activity for at least two years from the date of receipt. Once thawed, an aliquot can be kept at 4°C for up to one month for routine use; do not refreeze. If you observe increased background or declining signal over time, prepare a fresh aliquot. For long-term storage beyond two years, keep vials at minus 80°C, though this is rarely necessary for active labs.

What sample preparation steps are critical for detecting secreted Kallikrein-12?

Because KLK12 is a secreted serine protease, it partitions into culture supernatants or interstitial fluid rather than remaining cell-associated. For conditioned media, switch cells to serum-free or low-serum medium 24-48 hours before harvest to avoid interference from bovine serum proteases; concentrate the medium 10- to 20-fold using a 10-kDa cutoff centrifugal filter. For tissue lysates, homogenize in buffer containing protease inhibitors (but note that serine protease inhibitors like PMSF or aprotinin may also inhibit KLK12 activity if you plan functional assays). Include both extracellular and intracellular fractions if analyzing secretion kinetics. Fresh or snap-frozen samples yield better results than those subjected to multiple freeze-thaw cycles.