Anti-Kallikrein-10 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight band should I expect for Kallikrein-10 on Western blot?

Kallikrein-10 is synthesized as a 276-amino acid preproenzyme with a predicted molecular weight of approximately 31 kDa. On Western blot, you will typically observe the mature processed form around 28-30 kDa after signal peptide cleavage. Depending on your sample source and cell type, you may also detect the proform. Glycosylation is minimal but can shift the apparent MW slightly in certain expression systems. If you see multiple bands, consider that KLK10 can undergo autocatalytic processing. Use recombinant KLK10 as a positive control to confirm the expected migration pattern in your gel system.

Is this Kallikrein-10 antibody suitable for detecting downregulation in breast cancer samples?

Yes, this antibody is appropriate for that application. KLK10 was originally identified through differential display as substantially downregulated in breast tumor tissue compared to normal epithelium, making it relevant for tumor-suppressor studies. For Western blot, start at 1:1000 dilution with 20-50 µg total protein from lysates of matched normal and tumor samples. Because KLK10 is a secreted protein, you may also detect it in conditioned media from cultured cells. Include a positive control such as normal breast epithelial cell lysate or recombinant KLK10 to verify antibody performance, especially if tumor expression is near the detection limit.

Will this antibody cross-react with other kallikrein family members?

The kallikrein family comprises 15 closely related serine proteases encoded on chromosome 19q13.4, so cross-reactivity is a legitimate concern. This rabbit polyclonal was raised against human KLK10, and while the immunogen selection aims for specificity, sequence homology among kallikreins means you should validate specificity in your system. Run lysates known to express other kallikreins alongside KLK10-positive controls. If you observe unexpected bands, consider that KLK6 and KLK8 share structural motifs with KLK10. Peptide competition assays or siRNA knockdown can confirm band identity. The antibody has validated human reactivity; cross-reactivity with other family members has not been systematically ruled out.

Does this antibody work in mouse samples for Klk10 detection?

Mouse is listed as potential cross-reactivity, meaning sequence homology suggests the antibody may recognize mouse Klk10, but this has not been formally validated. Mouse Klk10 shares significant sequence identity with human KLK10 in conserved catalytic regions, which increases the likelihood of recognition. If you plan to use mouse tissue or cell lysates, we recommend empirical testing starting at 1:500 to 1:1000 dilution with a known Klk10-expressing mouse tissue as a positive control. Include a mouse sample known to lack Klk10 expression if available, or consider knockdown validation to confirm specificity before investing in a full experimental series.

What is the recommended starting dilution for IHC with this Kallikrein-10 antibody?

While the antibody has been validated for immunohistochemistry, the optimal dilution depends on your tissue type, fixation method, and detection system. For formalin-fixed paraffin-embedded human tissues, a reasonable starting range is 1:100 to 1:200. Antigen retrieval is typically required; citrate buffer pH 6.0 with heat-induced epitope retrieval is a standard starting method for kallikrein antibodies. Because KLK10 is secreted, you may observe both intracellular staining in producing cells and extracellular signal. Normal breast or prostate epithelium serve as useful positive-control tissues. Titre the antibody on your specific tissue and fixation protocol to balance signal and background.

Can I detect secreted Kallikrein-10 in cell culture supernatants by Western blot?

Yes, KLK10 is a secreted serine protease and can be detected in conditioned media. Concentrate culture supernatants 10- to 20-fold using centrifugal filters or precipitation methods before loading, as secreted protein concentrations are typically lower than intracellular levels. Use serum-free or low-serum media to reduce background from serum proteases. Be aware that secreted KLK10 may undergo autocatalytic processing or cleavage by other proteases in the medium, potentially yielding multiple bands. Include protease inhibitors during sample collection if you want to preserve the proenzyme form. The 1:1000 Western blot dilution should work well with concentrated supernatants; adjust based on expression level in your cell line.

How should I store this Kallikrein-10 antibody to maintain activity?

Store the antibody at -20°C in small aliquots to avoid repeated freeze-thaw cycles, which can reduce titer and increase aggregation. If you plan to use the antibody frequently over a short period, a working aliquot can be kept at 4°C for up to one month. Do not store diluted antibody; prepare fresh working dilutions in your blocking buffer for each experiment. Rabbit polyclonal antibodies are generally stable for at least two years at -20°C when handled properly. Sodium azide at 0.02-0.05% can be added as a preservative for 4°C storage if your downstream application permits it. Avoid prolonged exposure to room temperature and ensure the tube is tightly sealed to prevent evaporation and concentration changes.

What are appropriate positive and negative controls for Kallikrein-10 Western blots?

For positive controls, use normal breast or prostate epithelial cell lysates, as KLK10 is expressed in these tissues. Recombinant human KLK10 protein provides the most definitive control and confirms expected molecular weight. For negative controls, many breast and prostate cancer cell lines show downregulated or absent KLK10 expression and can serve as low-expression comparators. Alternatively, use lysate from a cell type not known to express KLK10, or employ siRNA knockdown in a KLK10-expressing line. Pre-incubating the antibody with blocking peptide corresponding to the immunogen, if available, offers an additional specificity control. Always include a loading control such as actin or tubulin to normalize protein input.