Anti-Kallikrein-5 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for KLK5 on a Western blot?

Kallikrein-5 migrates at approximately 33 kDa on Western blot, corresponding to the mature secreted form after cleavage of the N-terminal signal peptide from the 293-amino acid precursor. You may also observe a band around 28 kDa representing the fully activated form following propeptide removal. Some samples show additional higher-molecular-weight species, particularly in conditioned media, which can reflect glycosylation variants or complexes with endogenous inhibitors. Run positive controls from keratinocyte lysates or recombinant KLK5 to confirm the expected pattern in your system.

Does this antibody distinguish KLK5 from other kallikrein family members?

This polyclonal antibody was raised against a KLK5-specific immunogen and validated for human KLK5 detection. Given the high sequence homology across the 15-member kallikrein family, particularly within the catalytic domain, we recommend confirming specificity in your experimental context by running lysates with known expression of related kallikreins or using siRNA knockdown controls. Cross-reactivity with KLK7, which shares approximately 50 percent sequence identity and co-localizes in skin, has not been systematically ruled out. Validate with recombinant KLK5 alongside your samples for the most rigorous interpretation.

What is the recommended starting dilution for Western blot with this KLK5 antibody?

We recommend starting at 1:1000 dilution for Western blot applications with standard chemiluminescent detection. This dilution was validated using human keratinocyte lysates and recombinant KLK5. Optimal dilution may vary depending on KLK5 expression level in your sample type; skin-derived samples typically yield robust signal, whereas other tissues or cell lines with lower endogenous expression may require titration down to 1:500. Always include a positive control lysate and titre the antibody against your specific sample matrix to establish the working dilution that gives the best signal-to-background ratio in your hands.

Will this antibody detect mouse or rat Kallikrein-5 in my samples?

Mouse and rat cross-reactivity is predicted based on sequence homology but has not been experimentally validated. Human KLK5 shares approximately 70 percent amino acid identity with rodent orthologs, suggesting the antibody may recognize these species, but performance cannot be guaranteed. If working with mouse or rat samples, we recommend running side-by-side comparisons with human positive controls and considering orthogonal validation methods such as qPCR or an independent antibody. Some researchers have reported success with rodent skin lysates, but signal intensity and specificity should be confirmed in your experimental system before committing to large-scale experiments.

What sample preparation works best for detecting KLK5 in skin tissue lysates?

KLK5 is a secreted protease abundant in the stratum corneum, so mechanical disruption is critical for efficient extraction. Homogenize fresh-frozen or snap-frozen skin samples in RIPA or similar lysis buffer containing protease inhibitor cocktail; avoid repeated freeze-thaw cycles that degrade proteases. For isolated epidermis, dispase or trypsin separation followed by lysis yields cleaner results than whole-thickness skin. KLK5 is also readily detected in keratinocyte-conditioned medium; concentrate serum-free medium by ultrafiltration or precipitation if expression is low. Always include protease inhibitors during lysis to prevent auto-activation and degradation of both KLK5 and substrate proteins.

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

Yes, the antibody is validated for immunohistochemistry and should work on formalin-fixed paraffin-embedded skin tissue. Antigen retrieval is typically required; citrate buffer pH 6.0 with heat-induced epitope retrieval is a common starting point for kallikrein detection in skin sections. KLK5 localizes predominantly to the granular and cornified layers of the epidermis, so expect strong apical staining in normal skin. Titre the antibody starting at approximately 1:200 dilution and optimize retrieval conditions and incubation time with positive-control skin sections. Block endogenous peroxidase and use appropriate secondary reagents to minimize background in the keratinized layers.

How should I store this antibody and what is the expected shelf life?

Store the antibody at –20°C in single-use aliquots to avoid repeated freeze-thaw cycles, which can reduce titer and increase aggregation. The 100 µg format is sufficient for multiple experiments; aliquot 10-20 µL portions immediately upon receipt. Once thawed, the working aliquot can be held at 4°C with 0.02 percent sodium azide for up to one month, but long-term storage should remain frozen. Under these conditions, polyclonal antibodies typically retain activity for at least two years from manufacture date. Avoid storing diluted antibody in working solutions for extended periods; prepare fresh dilutions in blocking buffer for each experiment to maintain optimal performance.

What are common positive and negative controls for KLK5 antibody validation?

Human primary keratinocytes or HaCaT cells are standard positive controls; KLK5 expression increases upon calcium-induced differentiation, so compare low-calcium versus high-calcium conditions. Recombinant human KLK5 protein serves as a definitive positive control for molecular weight confirmation on Western blot. For negative controls, use tissues with minimal KLK5 expression such as liver, brain, or muscle lysates, or employ siRNA knockdown in keratinocyte cultures. In IHC, compare epidermis to dermis within the same section; dermis should show minimal to no signal. Pre-absorption with recombinant KLK5 can further confirm antibody specificity if cross-reactivity is a concern.