搜索

Chromogenic Substrate Assay for Determining the Activity of Plasma Kallikrein
显色底物分析测定血浆激肽释放酶活性   

下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

The activation of the intrinsic pathway takes place at negatively charged surfaces, such as bacteria, and involves activation of cogulation Factor XII, which then leads to the activation of plasma kallikrein (PK) and coagulation Factor XI. To determine the PK activity on bacterial surfaces, bacteria were pre-incubated with peptides, followed by incubation with plasma, and the effect of peptide was recorded by measuring the PK activity.

Materials and Reagents

  1. Specific bacteria [e.g. Escherichia coli (E. coli), ATCC, catalog number: 25922 Pseudomonas aeruginosa (P. aeruginosa), ATCC, catalog number: 27853 )]
  2. 3% Todd-Hewitt (TH) broth
  3. 3% TH agar plates
  4. Human citrated plasma (fresh)
  5. 50 mM Tris/HCl (pH 7.4, sterile)
  6. 50 mM ZnCl2 in H2O (sterile)
  7. Chromogenic substrate S-2302 (Chromogenix, catalog number: S820340 ) (4 mM in H2O)
  8. DAPTTIN TC (Technoclone, catalog number: 5035060 )
  9. Peptides (e.g. antimicrobial peptide LL-37) or other agents to be tested

Equipment

  1. Microcentrifuge tube
  2. BD vacutainer® plus blood collection tubes (BD, catalog number: 364305 )
  3. 96-well flat bottom plates
  4. Centrifuge for microcentrifuge tubes
  5. ELISA reader (A = 405 nm)
  6. Incubator 37 °C

Procedure

  1.   Assay with bacteria
         Day 1
    1. Grow bacteria overnight in fresh 10 ml 3% TH medium, at 37 °C and shaking with 200 rpm.
      Day 2
    2. In a fresh 10 ml 3% TH medium add 100 μl of overnight culture bacteria and subculture bacteria to mid-exponential phase (OD620 ~ 0.5), at 37 °C and 200 rpm.
    3. Centrifuge bacteria (10 min, 5,000 rpm) and remove the supernatant.
    4. Wash bacterial pellet twice with 10 ml of 50 mM Tris/HCl (pH 7.4).
    5. Resuspend bacterial pellet in 50 mM Tris/HCl (pH 7.4) + 50 µM ZnCl2 to a final concentration of 2 x 109 cfu/ml.
      Note: To get 2 x 109 cfu/ml, growth curves for the specific bacteria are necessary in order to know how much volume is needed for resuspention, before doing the experiment.
      Take 100 µl of bacteria and incubate with 10 µl of the test agent or buffer (positive control) for 60 sec at RT.
      Notes:
      1.  Negative controls contain only 100 µl bacteria mixed with 100 µl 50 mM Tris/HCl (pH 7.4) instead of plasma.
      2. Incubation times and amount of substrate might need adjustements according to the specific bacterium tested (Papareddy et al., 2013).
    6. Add 100 µl citrate plasma to the samples.
    7. Incubated 35 min at 37 °C on rotation 200 rpm.
    8. Spin down the bacteria (6 min, 5,000 rpm) and wash the bacterial pellets once with 50 mM Tris/HCl (pH 7.4) (100 µl/sample).
    9. Resuspend in 100 µl of 50 mM Tris/HCl (pH 7.4) + 50 µM ZnCl2 buffer containing 2 mM of the chromogenic substrate S-2302 (mix well).
    10. Incubate samples for 30-60 min at 37 °C (check yellow colour development in positive control, an indication of chromophore cleavage and dissociated from the enzyme), to determine incubation time.
    11. Centrifuge samples (6 min, 5,000 rpm) and transfer the supernatants to a 96 well plate.
    12. Measure absorbance at 405 nm. Values are presented in relation to the positive control (defined as 100%).

  2.  Assay without bacteria
    1. Incubate 10 µl of your desired agent with 100 µl of Dapttin for 60 sec at RT.
    2. Add 100 µl of human citrate plasma and incubate samples for 3 min at room temperature.
    3. Centrifuge the pellet (6 min, 5,000 rpm) and wash twice in 50 mM Tris/HCl (pH 7.4) before suspension in 100 µl 50 mM Tris/HCl (pH 7.4) + 50 µM ZnCl2 buffer containing 2 mM of the chromogenic substrate S-2302.
    4. Incubate for 30 min at RT.
    5. Centrifuge samples.
    6. Measure absorbance of the supernatant at 405 nm. Values are presented in relation to the positive control (defined as 100%).
      Notes:
      1. Regarding negative controls, add 100 µl of 50 mM Tris/HCl (pH 7.4) instead of adding 100 µl of human citrate plasma at step B2. But, everything else is the same from steps B1-6.
      2. Incubation time for the cleavge of substrate and subsequent colour development may differ. If the development of colour is to low, concentration of the substrate can be increased.

Acknowledgments

This protocol is adapted from Papareddy et al. (2013).

References

  1. Papareddy, P., Kalle, M., Sorensen, O. E., Malmsten, M., Morgelin, M. and Schmidtchen, A. (2013). The TFPI-2 derived peptide EDC34 improves outcome of gram-negative sepsis. PLoS Pathog 9(12): e1003803.

简介

内在途径的活化发生在带负电荷的表面,例如细菌,并且包括凝血因子XII的激活,其然后导致血浆激肽释放酶(PK)和凝血因子XI的激活。 为了测定细菌表面上的PK活性,将细菌与肽预温育,然后与血浆孵育,通过测量PK活性记录肽的效果。

材料和试剂

  1. 特定细菌[例如 大肠杆菌(E.coli),ATCC,目录号:25922; < em>绿脓假单胞菌(铜绿假单胞菌),ATCC,目录号:27853)]
  2. 3%Todd-Hewitt(TH)肉汤
  3. 3%TH琼脂平板上
  4. 人类柠檬酸血浆(新鲜)
  5. 50mM Tris/HCl(pH7.4,无菌)
  6. 50mM ZnCl 2在H 2 O(无菌)中的溶液
  7. 显色底物S-2302(Chromogenix,目录号:S820340)(4mM,在H 2 O中)
  8. DAPTTIN TC(Technoclone,目录号:5035060)
  9. 肽(例如抗微生物肽LL-37)或其他待测试剂

设备

  1. 微量离心管
  2. BD vacutainer ®加采血管(BD,目录号:364305)
  3. 96孔平底板
  4. 离心机用微量离心管
  5. ELISA读数器(A = 405nm)
  6. 培养箱37℃

程序

  1.   用细菌测试
         第1天
    1. 在新鲜的10ml 3%TH培养基中,在37℃下,以200rpm振荡,使细菌生长过夜 第2天
    2. 在新鲜的10ml 3%TH培养基中加入100μl过夜培养细菌 并在37℃将传代细菌传代至中指数期(OD <620>〜0.5)   和200rpm
    3. 离心细菌(10分钟,5,000rpm)并除去上清液
    4. 用10ml 50mM Tris/HCl(pH7.4)洗涤细菌沉淀两次
    5. 将细菌沉淀物在50mM Tris/HCl(pH 7.4)+50μMZnCl 2中重悬至终浓度为2×10 9 cfu/ml。
      注意:   为了获得2×10 9 cfu/ml,特定细菌的生长曲线 必要的,为了知道需要多少体积的重悬, 在进行实验之前。
      取100微升细菌,与10微升测试试剂或缓冲液(阳性对照)在室温孵育60秒。
      注意:
      1.  阴性对照仅含100μl细菌,与100μl50 mM Tris/HCl(pH 7.4)混合,而不是血浆。
      2. 孵育时间和底物量可能需要调整 根据所测试的特定细菌(Papareddy等人,2013年)。
    6. 向样品中加入100μl柠檬酸盐血浆
    7. 在37℃下在200rpm转速下孵育35分钟
    8. 旋转细菌(6分钟,5,000 rpm),并清洗细菌 用50mM Tris/HCl(pH 7.4)(100μl/样品)沉淀一次
    9. 重悬于100μl50mM Tris/HCl(pH 7.4)+50μMZnCl 2缓冲液 含有2mM发色底物S-2302(混合好)
    10. 在37°C孵育样品30-60分钟(检查黄色 在阳性对照中的发展,发色团切割的指示 并与酶解离),以确定孵育时间
    11. 离心样品(6分钟,5000rpm),并将上清液转移到96孔板中
    12. 测量405nm处的吸光度。 值相对于阳性对照(定义为100%)呈现。

  2.  无细菌测定
    1. 孵育10微升所需的代理与100微升Dapttin在室温60秒
    2. 加入100微升人类柠檬酸盐血浆,并在室温下孵育样品3分钟
    3. 离心沉淀(6分钟,5,000 rpm),并在50 mM中洗涤两次 Tris/HCl(pH7.4),然后悬浮于100μl50mM Tris/HCl(pH7.4)+中 50μM含有2mM发色底物S-2302的ZnCl 2缓冲液。
    4. 在室温下孵育30分钟。
    5. 离心样品。
    6. 测量上清液在405nm的吸光度。 值呈现 相对于阳性对照(定义为100%) 注意:
      1. 对于阴性对照,加入100μl的50mM Tris/HCl(pH 7.4) 而不是在步骤B2中加入100μl人柠檬酸盐血浆。 但, 其他一切都与步骤B1-6相同。
      2. 孵育时间 用于底物的裂解和随后的显色 不同。 如果颜色的发展是低,浓度 底物可以增加。

致谢

该协议改编自Papareddy等人(2013)。

参考文献

  1. Papareddy,P.,Kalle,M.,Sorensen,O.E.,Malmsten,M.,Morgelin,M。和Schmidtchen,A。(2013)。 TFPI-2衍生肽EDC34可改善革兰氏阴性脓毒症的结果。 PLoS Pathog 9(12):e1003803。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Papareddy, P., Kalle, M. and Schmidtchen, A. (2014). Chromogenic Substrate Assay for Determining the Activity of Plasma Kallikrein. Bio-protocol 4(19): e1246. DOI: 10.21769/BioProtoc.1246.
  2. Papareddy, P., Kalle, M., Sorensen, O. E., Malmsten, M., Morgelin, M. and Schmidtchen, A. (2013). The TFPI-2 derived peptide EDC34 improves outcome of gram-negative sepsis. PLoS Pathog 9(12): e1003803.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。