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
发布: 2014年10月05日第4卷第19期 DOI: 10.21769/BioProtoc.1246 浏览次数: 9571
评审: Fanglian HeAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Dec 2013
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
- Specific bacteria [e.g. Escherichia coli (E. coli), ATCC, catalog number: 25922 ; Pseudomonas aeruginosa (P. aeruginosa), ATCC, catalog number: 27853 )]
- 3% Todd-Hewitt (TH) broth
- 3% TH agar plates
- Human citrated plasma (fresh)
- 50 mM Tris/HCl (pH 7.4, sterile)
- 50 mM ZnCl2 in H2O (sterile)
- Chromogenic substrate S-2302 (Chromogenix, catalog number: S820340 ) (4 mM in H2O)
- DAPTTIN TC (Technoclone, catalog number: 5035060 )
- Peptides (e.g. antimicrobial peptide LL-37) or other agents to be tested
Equipment
- Microcentrifuge tube
- BD vacutainer® plus blood collection tubes (BD, catalog number: 364305 )
- 96-well flat bottom plates
- Centrifuge for microcentrifuge tubes
- ELISA reader (A = 405 nm)
- Incubator 37 °C
Procedure
文章信息
版权信息
© 2014 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
- 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.
- 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.
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分类
微生物学 > 微生物生物化学 > 蛋白质
免疫学 > 宿主防御 > 综合
生物化学 > 蛋白质 > 活性
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