| Application notes
(see key) | Recommended dilutions
SDS-PAGE: Use at an assay dependent dilution. Processed Kallikrein 5 has an apparent mass of 32-34 kDa on reduced SDS PAGE gels. WB: 1/1000 when using colorimetric substrates such as BCIP/NBT and 1/5000 for chemiluminescent substrates. Predicted molecular weight: 32 kDa. Dilution optimised using Chromogenic detection. A range of different sized bands can be seen for Kallikrein 5 in different tissues and in cell culture media, ranging from 16-32 kDa and likely representing different proteolytically processed forms, but perhaps also the different splice variants. Not yet tested in other applications. Optimal dilutions/concentrations should be determined by the end user. |
| Relevance | The human tissue Kallikrein gene family encodes 15 serine proteases. All Kallikreins share structural similarities including cysteine residues, a catalytic triad of His, Asp, and Ser residues, typically five coding exons, and varied intron phases. Kallikreins are predominantly secreted as inactive zymogens prior to activation by cleavage of an N terminal peptide, and all function extracellularly. Kallikreins can be activated autocatalytically, via other Kallikreins, or additional proteases. While structurally similar, Kallikrein family members have distinct functions and have key roles in many physiological and pathological processes. Many human tissue Kallikreins also show promise as cancer biomarkers, which may facilitate earlier detection and characterization of many forms of cancer.
Kallikrein 5 was originally described from the skin as an enzyme involved in turnover of the stratum corneum, and later cloned from human EST libraries as a Kallikrein like enzyme. The skin is known to contain trypsin like and chymotrypsin like proteinases of 32 kDa and 25 kDa respectively, and these were determined to be hK5 and hK7. Kallikrein 5 is found is greatest abundance in the skin, and also in the brain, with lesser signal in the kidney, lung, mammary gland, and testis. Human breast milk is reported to contain high levels of Kallikrein 5. Less stringent PCR detects hK5 in many tissues, and analysis of cell lines by Western blot shows that many cell lines make hK5.
Kallikrein 5 message is induced by estrogens in breast tumor cells, and hK5 has been reported to be elevated in ovarian cancer, but down regulated in prostate tumors, and kallikrein 5 has been reported to be elevated is sera of ovarian cancer patients Different splice variants of hK5 have also been implicated as markers in ovarian cancer. A 269 amino acid form with a shorter carboxyterminus that lacks the catalytic serine has been reported, as well as a 253 amino acid form using a different start site and containing a deletion that removes the catalytic cysteine. Endogenous inhibitors include kallistatin, protein C inhibitor and a1 proteinase inhibitor, although Kallikrein 5 can be found complexed to a number of different proteinase inhibitors. Pre-pro-kallikrein-5 has the 29 amino acid signal sequence that is removed before secretion, and the Pro-kallikrein-5 is activated to Kallikrein 5 by removal of the 37 amino acid propeptide domain. The full length hK5 sequence codes for a protein of 293 amino acids, with predicted mass of 32 kDa, and a very basic pI of 9.45, and the processed hK5 has an apparent mass of 32-34 kDa on reduced SDS PAGE gels. A range of different |
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