Anti-Kallikrein-8 Rabbit Polyclonal Antibody

Frequently Asked Questions

What is the expected molecular weight for Kallikrein-8 on Western blot?

Kallikrein-8 migrates at approximately 28–30 kDa on reducing SDS-PAGE, corresponding to the mature secreted protease after signal peptide cleavage. The full-length transcript encodes 260 amino acids, but the signal sequence is removed during secretion. You may observe a slightly higher apparent molecular weight depending on glycosylation state, as KLK8 contains potential N-glycosylation sites. In some tissue lysates, you might detect both pro-enzyme and active forms. Use recombinant KLK8 or hippocampal lysate as a positive control to confirm the expected band.

What starting dilution should I use for Kallikrein-8 Western blot?

We recommend starting at 1:1000 dilution for Western blot with standard chemiluminescent detection. This polyclonal antibody performs well at that concentration with 20–50 µg total protein per lane. If your tissue or cell line expresses KLK8 at lower levels—common outside hippocampus, skin, and reproductive tissues—you may need to concentrate your sample or increase antibody to 1:500. Titre against your specific lysate, as expression varies widely by cell type and activation state. Overnight incubation at 4°C often improves signal over one-hour room-temperature blocking.

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

The tissue kallikrein family comprises 15 closely related serine proteases with significant sequence homology, so cross-reactivity is a legitimate concern. This rabbit polyclonal was raised against a Kallikrein-8-specific region to minimize off-target binding, but we recommend including lysates known to express other kallikreins—such as prostate tissue rich in KLK2 and KLK3—as specificity controls. Validated reactivity is confirmed for human KLK8; cross-reaction with KLK5, KLK6, or KLK7 has not been systematically tested. Pre-absorption with recombinant KLK6 can help assess specificity if you work in skin or CNS samples.

Can I use this Kallikrein-8 antibody on mouse or rat brain tissue?

Mouse and rat reactivity are predicted based on sequence homology—human and rodent KLK8 share approximately 70–75 percent identity in the immunogenic region—but we have not formally validated these species. If you are working with mouse or rat hippocampus, start with the recommended 1:1000 dilution for Western blot and include a human tissue or recombinant KLK8 positive control on the same membrane. Some researchers report success with rodent brain lysates, but signal intensity may be lower. For IHC on rodent sections, empirical titration is essential.

What positive control tissue should I use for Kallikrein-8 experiments?

Hippocampal lysate is the gold-standard positive control for KLK8, as the protease is highly expressed in pyramidal neurons and plays a role in synaptic plasticity. Human skin, particularly epidermis, is another reliable source. Recombinant human KLK8 offers the cleanest single-band control and confirms antibody specificity without interference from other proteases. Ovarian tissue also expresses KLK8, though levels vary across the menstrual cycle. Negative controls should include tissues with negligible KLK8 expression, such as liver or skeletal muscle, to assess background binding.

How should I prepare samples to detect secreted Kallikrein-8?

Because KLK8 is a secreted protease, conditioned medium from cultured cells or cerebrospinal fluid can be excellent sources, but you must concentrate samples before Western blot. Use centrifugal filters with a 10-kDa cutoff or TCA precipitation to achieve 10- to 20-fold concentration. Include protease inhibitors—particularly serine protease inhibitors like AEBSF or PMSF—in collection buffers, since active KLK8 can degrade itself and other substrates. For tissue lysates, homogenize in RIPA buffer with inhibitor cocktail and clarify by high-speed centrifugation to remove insoluble material that may trap secreted protein.

What is the storage stability of this Kallikrein-8 antibody after reconstitution?

The lyophilized antibody is stable at –20°C for at least two years as supplied. After reconstitution in sterile PBS or 50 percent glycerol, store aliquots at –20°C and avoid repeated freeze-thaw cycles, which can aggregate immunoglobulins and reduce titer. For frequent use, keep a working aliquot at 4°C for up to one month; supplement with 0.02 percent sodium azide if you prefer liquid storage without glycerol. We have not observed significant loss of activity within six months at –20°C in glycerol. Do not store diluted antibody in blocking buffer for more than one week.

Does Kallikrein-8 require special reducing conditions for Western blot detection?

KLK8 is a single-chain serine protease and does not require reducing conditions for activity, but standard reducing SDS-PAGE with beta-mercaptoethanol or DTT is recommended for Western blot to denature the protein fully and yield a single sharp band near 28–30 kDa. Under non-reducing conditions, you may see disulfide-linked dimers or aggregates, particularly if samples contain active protease that has cross-linked substrates. For most applications, treat samples with Laemmli buffer plus reducing agent and boil for five minutes before loading. This antibody recognizes the denatured, reduced form reliably.