Anti-Kallikrein-4 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for Kallikrein-4 on Western blot?

Kallikrein-4 has a predicted molecular weight of approximately 28 kDa based on its 254-amino acid sequence. However, the protein is synthesized as a preproenzyme with a signal peptide that is cleaved during secretion, so the mature, active form typically migrates around 25-27 kDa. In some tissue lysates, particularly from prostate or dental tissue, you may observe bands near 30 kDa representing the pro-form. Post-translational modifications including glycosylation have been reported in certain contexts, which can shift apparent molecular weight. Always include a positive control lysate from KLK4-expressing tissue to confirm the expected pattern.

What is the recommended starting dilution for this Kallikrein-4 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 with standard chemiluminescent detection systems. If your sample has low KLK4 expression or you are working with secreted fractions where the protease may be dilute, you may need to increase antibody concentration to 1:500. Conversely, for overexpression lysates or recombinant protein, 1:2000 may provide cleaner signal. Titrate against your specific lysate type, as ameloblast-derived samples will have substantially higher KLK4 than most other tissue sources.

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

Kallikrein-4 shares sequence homology with fourteen other human kallikrein proteases, particularly KLK5, KLK6, and KLK7. This polyclonal was raised against a relatively selective epitope region to minimize cross-reactivity, but kallikrein family members can co-migrate near 25-30 kDa on Western blots. For samples with multiple kallikreins present, such as prostate or skin tissue, consider running a KLK4 knockout or knockdown lysate as a specificity control. The antibody has been validated against recombinant KLK4 and in tissues where KLK4 is the predominant kallikrein, such as maturation-stage dental epithelium.

Which species can I use this Kallikrein-4 antibody with?

This antibody is validated for human KLK4 detection. Cross-reactivity with monkey, dog, and pig is predicted based on epitope sequence conservation but has not been experimentally confirmed in-house. If you are working with rodent samples, note that mouse and rat KLK4 orthologs show lower sequence identity in several epitope regions, and we cannot guarantee reliable detection. For non-human work, we recommend confirming specificity by including a lysate spike with recombinant human KLK4 or running parallel samples from a validated human source to benchmark signal intensity and band pattern.

What sample types and lysis conditions work best for detecting Kallikrein-4?

Because KLK4 is a secreted serine protease, it is enriched in conditioned media, saliva, and extracellular fractions rather than whole-cell lysates. For tissue samples, ameloblast-rich dental organ extracts during enamel maturation are ideal positive controls. Prostate tissue and seminal plasma also contain detectable KLK4. Use standard RIPA or NP-40 lysis buffers; avoid reducing agents during sample preparation if you need to preserve disulfide-dependent epitopes, though this antibody typically performs well under standard reducing SDS-PAGE conditions. Store lysates at -80°C and minimize freeze-thaw cycles, as proteolytic activity can degrade target.

Can this antibody detect both pro-KLK4 and the active mature form?

This polyclonal antibody recognizes epitopes present in both the pro-enzyme and mature active forms of Kallikrein-4. The pro-form includes an N-terminal activation peptide that is cleaved by trypsin-like proteases to generate the active enzyme. Depending on tissue context and processing state, you may see a doublet or two distinct bands on Western blot corresponding to these forms. In ameloblast secretions or extracellular samples with active proteolytic processing, the mature form typically predominates. If distinguishing activation state is critical to your experiment, consider running samples with and without exogenous protease inhibitors to assess cleavage.

What are the proper storage conditions for this Kallikrein-4 antibody?

Store the antibody at -20°C in the supplied glycerol-containing buffer. Avoid repeated freeze-thaw cycles, which can reduce antibody titre and increase background. For routine use, we recommend preparing single-use aliquots immediately upon receipt: thaw the stock once, dispense 10-20 µL volumes, and refreeze. Working aliquots diluted in blocking buffer with 0.02 percent sodium azide can be stored at 4°C for up to one week, though fresh dilutions generally provide more consistent signal. Do not store diluted antibody long-term or expose the stock vial to temperatures above 4°C for extended periods.

What positive and negative controls should I use when validating Kallikrein-4 detection?

For positive controls, recombinant human KLK4 protein is ideal if available. Biologically, maturation-stage ameloblast lysates or conditioned media from ameloblast cell lines provide robust signal. Prostate tissue lysates or LNCaP prostate cancer cell lysates are more accessible alternatives, though KLK4 expression is lower than in dental tissue. Negative controls should include tissues with minimal or absent KLK4 expression, such as liver, skeletal muscle, or lymphocytes. Knockout cell lines or siRNA-treated samples targeting KLK4 serve as specificity controls to confirm observed bands are not from cross-reactive kallikreins or non-specific binding.