来源于溶组织梭菌的梭菌蛋白酶是一种蛋白酶，可在精氨酸残基的羧基端切割肽键，也称为 Arg‑C 内肽酶。作为蛋白水解酶，可通过组织解离用于多种类型细胞的分离。
梭菌蛋白酶存在两种形式：氧化型（天然态）与还原型（激活态），二者酶活性存在差异。
产品描述
梭菌蛋白酶 NB 经过色谱高度纯化，基本不含其他杂酶活性。
应用
梭菌蛋白酶 NB 可与胶原酶 NB 1, 优级品（货号 C278986）和 / 或中性蛋白酶 NB（货号 N279015）搭配使用，适用于从各类组织中分离细胞。
使用说明
1、概述
梭菌蛋白酶 NB与 胶原酶 NB 1, 优级品（货号 C278986）、中性蛋白酶 NB（货号 N279015）配合使用， 尤其适用于从人胰腺分离朗格汉斯胰岛，也可用于多种其他人源或动物源组织的细胞分离。天然态梭菌蛋白酶可使用还原剂（例如 DTT、半胱氨酸）激活，激活后酶活性升高。
2、组织解离
人胰腺朗格汉斯胰岛分离的推荐起始工作浓度：
胶原酶 NB 1, 优级品：15‑20 PZ U /g 组织
中性蛋白酶 NB：0.8‑1.5 DMC U /g 组织
梭菌蛋白酶 NB（天然态）：0.8‑1.8 U /g 组织
胶原酶中会含有微量梭菌蛋白酶，计算体系内梭菌蛋白酶终浓度时必须将该部分纳入考量。
总体而言，合适的梭菌蛋白酶用量取决于组织类型、组织来源以及分离实验方案。
3、储备液
用缓冲液溶解酶，配制梭菌蛋白酶 NB 储备液；酶溶液必须全程冰上保存。
梭菌蛋白酶活性依赖钙离子，体系中严禁存在钙螯合剂（如 EDTA）。
复溶后的梭菌蛋白酶 NB 可经 0.22 μm 滤膜过滤、分装，‑20 ℃保存；避免反复冻融，分装后样品可稳定保存 1 年。
进行 0.22 μm 过滤时，建议选用低蛋白吸附滤膜（如醋酸纤维素、PVDF、PES 材质）。
4、工作液
用缓冲液稀释储备液，配制实验所需浓度的工作液；工作液在使用前必须全程冰上放置。
若将梭菌蛋白酶 NB 溶液与胶原酶 NB、中性蛋白酶 NB 溶液混合，混合液需立即使用。
5、酶的失活与抑制剂
可通过降温或者稀释酶溶液，减缓组织解离反应进程。
梭菌蛋白酶抑制剂包含 EDTA 这类螯合剂，以及所有氧化剂。
6、重要提示
梭菌蛋白酶 NB仅可用于科研。
Clostripain from Clostridium histolyticum is a proteinase that cleaves peptide bonds on the carboxyl side of arginine. It is also known as Endoproteinase Arg‑C. As a proteolytic enzyme it can be used for isolation of various cell types by tissue dissociation.
Clostripain exists in two different forms, an oxidized (native condition) and a reduced (activated) form that differ in enzymatic activity.
Description
Clostripain NB is chromatographically highly purified and largely free from additional enzymatic activities.
Application
Clostripain NB is used in combination with Collagenase NB 1 Premium Grade (Cat. No. C278986) and/or Neutral Protease NB (Cat. No. N279015), suitable for cell isolation from various tissue types.
Instructions for use:
1. General
Clostripain NB is, in combination with Collagenase NB 1 Premium Grade and Neutral Protease NB, especially suited for isolation of islets of Langerhans from human pancreas, and also for cell isolation from several other human or animal tissues. The native clostripain can be activated with reducing agents (e.g. DTT, Cysteine) resulting in a higher activity of the enzyme.
2. Tissue dissociation
Recommended starting concentrations for isolation of islets of Langerhans from human pancreas:
Collagenase NB 1 Premium Grade: 15 – 20 PZ U/g tissue
Neutral Protease NB: 0.8 – 1.5 DMC U/g tissue
Clostripain NB, native state: 0.8 – 1.8 U/g tissue
Collagenase may contain traces of clostripain which have to be considered to calculate the final clostripain concentration.
In general, the appropriate clostripain concentration depends on tissue type and origin as well as on the isolation procedure.
3. Stock solution
A stock solution of Clostripain NB is prepared by dissolving the enzyme in buffer. The enzyme solution must be constantly stored on ice.
Since Clostripain depends on calcium, absolutely no calcium chelating agents (e.g. EDTA) should be present at all.
Reconstituted Clostripain NB can be 0.22 μm filtered, aliquoted and stored at -20 °C. Aliquots are stable for 1 year if repeated freezing and thawing is avoided.
For 0.22 μm filtration filters with low protein‑binding properties (e.g. cellulose acetate, PVDF, or PES) are recommended.
4. Working solution
To prepare a working solution, the stock solution is diluted with buffer to achieve the required clostripain concentration. The working solution must be constantly stored on ice until use.
If Clostripain NB solution is mixed with Collagenase NB and Neutral Protease NB solution, the blend should be used immediately.
5. Inactivation and inhibitors
The dissociation process can be reduced, e.g. by cooling down or dilution of the enzyme solution. Inhibitors of Clostripain include chelating agents like EDTA and all oxidizing agents.
6. Important note
Clostripain NB is intended for research use only.
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