Neutral Protease Dispase®

Description

Background

Neutral protease (Dispase®) is a non-mammalian animal origin free metallo, neutral protease. Its mild proteolytic action makes the enzyme especially suitable for the preparation of primary cells and secondary (subcultivation) in cell culture since it is gentle on cell membranes. This protease is also used as a secondary enzyme in cell isolation and tissue dissociation applications, commonly used with collagenase. Neutral protease is an extremely stable Zn-metalloendopeptidase that is produced by Paenibacillus polymyxa. It is involved in the generation of beta- and alpha-amylases from the large amylase precursor. Although related to trypsin, neutral protease is significantly less harmful to cells and can help prevent unwanted cell clumping without cell membrane damage after one hour incubations. Neutral protease (Dispase®) is ideal for researchers focused in Stem Cell and Biomarker Research.

Application Note

Neutral protease (Dispase®) is designed for primary cell isolation and tissue dissociation. It is commonly used to separate skin epidermis from dermis leaving intact epithelial sheets and stem cell, hepatocyte and other cell isolation applications. However, due to the diversity of the variables involved, exact isolation conditions should be determined empirically for each cell/tissue application. Expect a band approximately 36 kDa in size by western blot in the appropriate tissue or cell lysate. Specific conditions for reactivity should be optimized by the end user.

Purity/Specificity

Neutral protease (Dispase®) is purified and one unit releases one micromole of Folin positive amino acids, measured as tyrosine, at 37°C, pH 7.5, using casein as the substrate. The enzyme is known to contain 1g-atom of zinc per g-mol of purified enzyme. If this zinc component is removed by chelating agents such as EDTA or EGTA, an inactive apoenzyme is obtained. Calcium has been detected in the purified protein and is believed to play a role in maintaining the structure and configuration, and preventing autolysis. Neutral protease is a non-specific metalloprotease. It cleaves fibronectin, collagen IV, and to a lesser extent collagen I, but it does not cleave collagen V or laminin. It hydrolyzes N-terminal peptide bonds of non-polar amino acid residues and may preferentially attack denatured and intercellular proteins with exposed hydrophobic amino acid residues. It is believed to bind one zinc ion and four calcium ions per subunit. Unlike other Bacillus species that produce neutral, alkaline, or a mixture of both proteases, Paenibacillus polymyxa is one of three species that produces only a neutral protease.