Anti-Kallikrein-14 Rabbit Polyclonal Antibody

Frequently Asked Questions

What is the expected molecular weight for KLK14 on Western blot?

Human KLK14 (UniProt Q9P0G3) encodes a 254-amino-acid preproprotein with a predicted molecular weight of approximately 28 kDa. After signal peptide cleavage, the secreted proenzyme runs near 26 kDa. Active KLK14, following propeptide removal, migrates at approximately 23–24 kDa under reducing conditions. You may observe both pro- and mature forms depending on the tissue or cell-culture system. Glycosylation is not a major feature of KLK14, so the band pattern is typically simpler than for heavily modified proteases. Use conditioned media or extracellular fractions for optimal detection, as KLK14 is a secreted protein.

What starting dilution should I use for Kallikrein-14 Western blots?

We recommend beginning at 1:1000 for Western blot, which works well with standard chemiluminescence detection and 10–30 µg total protein per lane. If your sample has low KLK14 expression—common in non-prostatic tissues—consider 1:500 or enriching secreted proteins from conditioned medium by ultrafiltration or precipitation. Conversely, prostate tissue lysates or seminal plasma may require dilution to 1:2000 to avoid saturation. Always include a sample known to express KLK14 (e.g., LNCaP or PC-3 prostate cell lysate, or recombinant KLK14) as a positive control to confirm the expected 23–26 kDa band.

Does this antibody cross-react with other kallikreins on Western blot?

The tissue kallikrein family shares significant sequence homology, particularly within the catalytic domain. While this polyclonal was raised against a KLK14-specific region, some cross-reactivity with closely related kallikreins—especially KLK5, KLK7, and KLK13—can occur in complex lysates. To confirm specificity, compare blots from KLK14-expressing versus KLK14-null samples or use siRNA knockdown. If you see multiple bands in the 23–28 kDa range, peptide competition or recombinant KLK14 spiking can help identify the genuine KLK14 signal. Predicted cross-reactivity with mouse, monkey, and dog orthologs has not been experimentally validated; confirmation with species-matched positive controls is advised.

What sample types and lysis buffers work best for detecting KLK14?

Because KLK14 is secreted, conditioned cell-culture medium, seminal plasma, or skin-tape-strip extracts are ideal sample types. For tissue lysates, use RIPA or similar non-denaturing buffers that preserve soluble extracellular proteins; avoid harsh detergents that may aggregate proteases. Collect medium after 24–48 hours of serum-free culture and concentrate by ultrafiltration (10-kDa cutoff) to enrich KLK14. Include protease inhibitors (AEBSF, aprotinin) during lysis to prevent autocatalytic degradation, though KLK14 is relatively stable. Avoid high-salt extraction buffers used for nuclear proteins, which dilute extracellular components and reduce sensitivity.

Can I use this antibody for immunohistochemistry on skin or prostate sections?

Yes, Triple Point Biologics antibodies are validated for Western blot; IHC application data is in progress, and KLK14 is physiologically expressed in both prostate epithelium and the granular-to-cornified layers of epidermis. For paraffin sections, antigen retrieval with citrate buffer (pH 6.0) or Tris-EDTA (pH 9.0) at sub-boiling temperature typically enhances signal. KLK14 localizes to the extracellular space and cell surface, so expect diffuse staining around glandular lumens in prostate or intercellular staining in the upper epidermis. Include a KLK14-low tissue (e.g., liver) as a negative control and block endogenous peroxidase. Start at 1:200–1:500 dilution and optimize empirically.

Which human cell lines or tissues express KLK14 for positive controls?

Prostate-derived cell lines—LNCaP, PC-3, and DU145—express detectable KLK14, with LNCaP often showing the highest levels, particularly in conditioned medium. Primary prostate tissue, seminal plasma, and skin keratinocytes (especially differentiated cultures) are also reliable sources. Ovarian tissue and some ovarian cancer cell lines (e.g., OVCAR-3) express KLK14, though levels vary. For Western blot validation, recombinant human KLK14 (available from several vendors) provides an unambiguous positive control and confirms the expected molecular weight. Negative controls include tissues with minimal expression, such as liver, skeletal muscle, or brain homogenates.

What are the recommended storage conditions and shelf life for this antibody?

Store the antibody at –20 °C in single-use aliquots to avoid repeated freeze-thaw cycles, which can reduce titre and increase aggregation. The polyclonal is supplied in a stabilized buffer at 1 mg/mL (100 µg total); aliquot 10–20 µL portions immediately upon receipt. For short-term use (up to one month), 4 °C storage is acceptable, but long-term storage at –20 °C or –80 °C preserves activity for at least two years. Do not store diluted working solutions; prepare fresh in blocking buffer on the day of use. Avoid sodium azide concentrations above 0.02 percent if you plan to use the antibody for IHC with HRP detection.

Does KLK14 require activation, and will the antibody detect both pro- and active forms?

KLK14 is synthesized as an inactive zymogen (pro-KLK14) and requires proteolytic removal of an N-terminal propeptide for enzymatic activity. In seminal plasma and skin, this activation occurs through cleavage by other kallikreins (notably KLK3 and KLK5) or trypsin-like proteases. This polyclonal antibody is raised against a large fragment (residues 46–260, spanning catalytic domain), so it should recognize both the pro- (≈26 kDa) and mature (≈23–24 kDa) forms on Western blot. The ratio of pro- to active KLK14 varies by tissue and physiological context, providing useful information about local proteolytic cascades.