Worthington dissociating enzyme: detailed analysis of neutral protease (dispase)

Amigen Worthington Neutral Protease (Dispase):

Neutral protease (dispase) is a bacterial enzyme produced by Bacillus polymyxa that hydrolyzes N-terminal peptide bonds of non-J amino acid residues and is classified as an aminoendopeptidase.Its mild proteolytic action makes this enzyme particularly suitable for the separation of primary and secondary (subcultured) cells, as it preserves the integrity of the cell membrane.

Neutral proteases (dispases) are also often used as auxiliary enzymes along with collagenases and/or other proteases in many primary cell isolation and tissue dissociation applications.Neutral protease (Dispase) dissociates fibroblast-like cells more efficiently than epithelioid cells, so it is also used in differential isolation and culture applications.Other advantages are its non-mammalian (bacterial) origin and ability to be inhibited by EDTA.

 

Tissue isolation/primary cell isolation and cell harvesting are the main applications of enzymes in tissue culture, stem cell research and cell biology research.The goal of a cell isolation procedure is to maximize the yield of functionally viable isolated cells.There are many parameters that can affect the results.The choice of enzyme is an important parameter.The Worthington Tissue Isolation Guide summarizes how these enzymes accomplish the "routine" manipulation of tissue isolation and primary culture cell harvesting.

Amogene Worthington Neutral Protease (Dispase) Specificity:

Neutral protease is a non-specific metalloprotease.It cuts fibronectin, collagen IV and to a lesser extent collagen I, but it does not cut collagen V or laminin.It hydrolyzes N-terminal peptide bonds of non-J amino acid residues and may preferentially attack denatured and intercellular proteins with exposed hydrophobic amino acid residues.Each subunit is believed to bind one zinc ion and four calcium ions.Unlike other Bacillus species that produce neutral, alkaline or a mixture of both proteases, Paenibacillus polymyxa is one of three species that produces only neutral proteases (Fogarty and Griffin 1973).

Ai Meiji Worthington Neutral Protease (Dispase) Application:

Tissue disaggregation and subculture

Prevent unwanted cell aggregation

Preparation of cultured cells

Isolation of the intact epidermis from the dermis and separation of the intact epithelial layer from the culture medium (Kurt et al. 1989)

Harvest and transfer of normal, diploid cells and cell lines (Matsumura et. al 1975)

Gentle and complete detachment of epidermal cells (Kitano and Okada 1983)