Anti-Kallikrein-7 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight band should I expect for KLK7 on a Western blot?

Kallikrein-7 runs at approximately 28 kDa on reducing SDS-PAGE, corresponding to the mature secreted protease after cleavage of the signal peptide. You may observe a slightly higher band around 30-32 kDa representing the pro-enzyme form (pro-KLK7) depending on your sample source. In conditioned media from cultured keratinocytes or epidermal extracts, expect the mature 28 kDa species to predominate. Glycosylation is minimal, so band shifts due to post-translational modification are uncommon. If you see multiple lower bands, consider proteolytic degradation or autocatalytic cleavage during sample preparation.

What dilution should I start with for Western blot detection of KLK7?

We recommend starting at 1:1000 dilution for Western blot, which has been validated with this polyclonal antibody. For highly abundant samples such as stratum corneum extracts or conditioned medium from differentiating keratinocytes, you may achieve clean signal at 1:2000 or 1:5000. For lysates with low KLK7 expression, titrate down to 1:500. Always include a positive control lysate from human epidermis or HaCaT cells induced to differentiate. Incubate overnight at 4°C for optimal sensitivity, especially when working with samples where KLK7 is expressed at low to moderate levels.

Does this antibody detect mouse or rat KLK7 orthologs?

Cross-reactivity with mouse and rat Klk7 is predicted based on sequence homology but has not been formally validated. Human KLK7 shares approximately 70-75% identity with rodent orthologs, with conservation higher in the catalytic domain than in the N-terminal region. If you are working with mouse or rat samples, plan to empirically test the antibody starting at 1:500 dilution and include a human positive control for comparison. Primate KLK7 from monkey and chimpanzee is expected to cross-react well given the higher sequence identity, though validation data are not yet available.

Can I use this KLK7 antibody for immunohistochemistry on skin sections?

Yes, This antibody is validated for Western blot; immunohistochemistry/immunofluorescence validation is in progress. KLK7 localizes predominantly to the stratum granulosum and stratum corneum layers of human epidermis. For paraffin-embedded sections, antigen retrieval with citrate buffer (pH 6.0) at sub-boiling temperature for 10-20 minutes typically yields optimal staining. Start with a 1:100 to 1:200 dilution. KLK7 is secreted, so expect extracellular and pericellular staining rather than sharp cytoplasmic localization. Sebaceous glands and eccrine sweat ducts may also show positive staining. Frozen sections generally require less dilution of primary antibody due to better epitope preservation.

What is the best way to prepare keratinocyte lysates for KLK7 detection?

Because KLK7 is a secreted serine protease, you will find higher concentrations in conditioned culture medium than in cell lysates. For secreted KLK7, collect serum-free conditioned medium from differentiated keratinocytes and concentrate 10- to 20-fold using centrifugal filters (10 kDa cutoff). For whole-cell lysates, use RIPA buffer with protease inhibitor cocktail; note that intracellular KLK7 levels are low unless cells are highly differentiated. Avoid repeated freeze-thaw cycles, which can lead to autocatalytic degradation. Calcium-containing buffers may enhance KLK7 activity and accelerate degradation, so work quickly and keep samples cold.

Does KLK7 have splice variants or isoforms I should be aware of?

The predominant KLK7 transcript encodes a 277-amino acid preproprotein (UniProt P49862). Minor splice variants have been reported in the literature but are not well characterized and likely represent low-abundance transcripts. On Western blot, you should not expect multiple isoform bands under typical conditions. The signal peptide is cleaved during secretion, yielding the mature 28 kDa enzyme, and the propeptide is removed upon activation. If you observe additional bands, consider post-translational cleavage or degradation rather than alternative splicing. Most functional studies focus on the canonical secreted form, which is the species this antibody targets.

What positive and negative control tissues should I use for KLK7 experiments?

Human stratum corneum or full-thickness skin extracts serve as excellent positive controls due to high endogenous KLK7 expression in the upper epidermis. Cultured primary human keratinocytes differentiated with high calcium medium also express robust KLK7. For negative controls, use tissues with minimal to no KLK7 expression such as liver, brain, or skeletal muscle lysates. When validating specificity, consider peptide competition experiments using the immunogen peptide if available. Netherton syndrome patient samples, which have dysregulated KLK7 activity due to SPINK5 mutations, can provide pathophysiological context but show elevated rather than absent KLK7.

How should I store this KLK7 antibody to maintain long-term stability?

Store the antibody at -20°C in small aliquots to avoid repeated freeze-thaw cycles, which can reduce titer and increase aggregation. The antibody is supplied as a rabbit polyclonal in buffer that typically contains glycerol and a preservative. Once thawed, an aliquot can be kept at 4°C for up to one month for frequent use. Do not store diluted antibody; prepare working dilutions fresh in blocking buffer immediately before use. If you observe precipitate after thawing, centrifuge briefly at 10,000 x g before use. Maintained properly, polyclonal antibodies retain activity for several years when stored frozen.