Anti-Kallikrein-3 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for PSA/Kallikrein-3 on Western blot?

Mature PSA migrates at approximately 28–33 kDa on reducing SDS-PAGE, depending on its glycosylation state. The full-length 261-amino-acid precursor includes a 17-residue signal peptide and a 7-residue propeptide that are cleaved during secretion. Glycosylation at Asn-45 accounts for the diffuse band appearance and slight upward shift from the predicted 26 kDa mass. In prostate tissue lysates or seminal fluid, you may observe the mature active form; serum samples often contain PSA complexed with alpha-1-antichymotrypsin, which migrates around 80–90 kDa. Running a purified PSA standard alongside your samples helps confirm the band identity.

What is the recommended starting dilution for this Kallikrein-3 antibody in Western blot?

We recommend starting at 1:1000 dilution for Western blot applications with this rabbit polyclonal antibody. This dilution has been validated against human prostate lysates and recombinant PSA. Depending on your sample type and abundance of Kallikrein-3, you may need to adjust between 1:500 and 1:2000. Prostate cancer cell lines such as LNCaP express high levels of PSA and typically yield strong signal at 1:1000, whereas normal prostate tissue or serum samples may benefit from higher antibody concentration. Always include a PSA-negative control, such as HeLa or HEK293 lysate, to confirm specificity.

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

This polyclonal was raised against Kallikrein-3 and validated for human PSA specificity. The tissue kallikrein family comprises 15 serine proteases with significant sequence homology, so cross-reactivity is a legitimate concern. Based on our validation data, we have not observed cross-reactivity with the most closely related family members, KLK2 and KLK4, in Western blot at recommended dilutions. However, we recommend including recombinant controls or knockout lysates if your experimental system co-expresses multiple kallikreins. Specificity can be further confirmed by peptide competition assays using the immunogen sequence or by siRNA knockdown of KLK3 in your cell line.

Is this PSA antibody validated for non-human primate samples?

This antibody is validated for human Kallikrein-3 and predicted to cross-react with Old World monkey orthologs based on sequence homology. Rhesus and cynomolgus macaque PSA share approximately 88–90% amino acid identity with human KLK3, with conservation highest in the catalytic domain. We have not formally validated reactivity in primate samples, so if you are working with macaque prostate tissue or cell lines, plan to empirically test dilutions between 1:500 and 1:2000. Be aware that PSA expression in non-human primates is lower than in humans, so signal intensity may be reduced. Run a human prostate lysate as a positive control.

What positive and negative controls should I use for Kallikrein-3 Western blots?

LNCaP cells are the gold standard positive control for PSA expression; these androgen-responsive prostate adenocarcinoma cells secrete high levels of Kallikrein-3 into culture medium and show robust signal in whole-cell lysates. Recombinant human PSA is also commercially available and useful for confirming the expected molecular weight. For negative controls, use cell lines that do not express prostatic markers, such as HeLa, HEK293, or Jurkat. Including both lysate and conditioned medium from LNCaP can help you distinguish intracellular precursor from secreted mature PSA. If studying regulation, treat LNCaP with DHT to upregulate PSA or with antiandrogens to suppress it.

Can I use this antibody to detect PSA in human serum or plasma samples?

Yes, this antibody can detect PSA in serum, though Western blot is not the most sensitive method for clinical samples where PSA concentrations are typically nanogram-per-milliliter range. In serum, most PSA circulates bound to alpha-1-antichymotrypsin or alpha-2-macroglobulin, forming high-molecular-weight complexes. You may see bands at approximately 80–90 kDa in addition to or instead of free 28–33 kDa PSA. For detection, concentrate serum samples or use a larger loading volume. Start antibody dilution at 1:500 and expect weaker signal than from tissue lysates. ELISA or immunoassay platforms remain more quantitative for serum PSA measurement.

How should I store this Kallikrein-3 antibody and what is its shelf life?

Store the antibody at –20°C in the supplied glycerol-based storage buffer. Under these conditions, the polyclonal remains stable for at least 12 months from the date of receipt. Avoid repeated freeze-thaw cycles, which can aggregate IgG and reduce titer; if you use the antibody frequently, aliquot it into single-use volumes immediately upon arrival. For short-term use over a few weeks, the working aliquot can be held at 4°C with 0.02% sodium azide as preservative. Do not store diluted antibody in blocking buffer for extended periods. If you observe declining signal over time, re-validate dilution with a positive control lysate before troubleshooting other variables.

What sample preparation considerations are important for detecting Kallikrein-3?

PSA is a secreted protease, so sample choice matters. For endogenous expression, use prostate tissue lysates or androgen-responsive prostate cancer cell lines such as LNCaP or VCaP. Harvest cells in log-phase growth and include protease inhibitors during lysis to prevent autocatalysis, though mature PSA is relatively stable. If collecting conditioned medium, serum-free or low-serum conditions minimize interfering albumin bands. PSA secretion is androgen-regulated, so expect variability with hormonal treatments or between cell passages. For reducing gels, standard Laemmli buffer with beta-mercaptoethanol or DTT is appropriate. Non-reducing conditions may preserve PSA complexes with serpins, shifting the apparent molecular weight upward.