Measurement of Dipeptidylpeptidase Activity in vitro and in vivo
体外和体内二肽基肽酶活性测定
发布: 2017年03月20日第7卷第6期 DOI: 10.21769/BioProtoc.2184 浏览次数: 8519
评审: Ivan ZanoniMeenal SinhaYang Fu
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参见作者原研究论文
The authors used this protocol in:
Jul 2015
Abstract
Dipeptidylpeptidases (DPPs) are serine proteases, which cleave small proteins and peptides possessing a proline or an alanine in the second position of their N-terminus. Among the members of this family, dipeptidylpeptidase 4 (DPP4) is constitutively expressed in the extracellular space. DPP4 is found at the surface of many hematopoietic and non-hematopoietic cells and is also present in many biological fluids in a bioactive soluble form. DPP4 expression is modulated by inflammation, and measurements of its activity have been used as biomarker for disease. Here, we describe a method to evaluate the enzymatic activity of DPP4 in vitro and in vivo.
Keywords: Dipeptidylpeptidase 4 (二肽基肽酶4)
Enzyme (酶)
Tumor (肿瘤)
Plasma (血浆)
DPP4 inhibitor (DPP4抑制剂)
Background
The magnitude of the enzymatic activity of DPPs can be an indicator of inflammation; and an important pharmacodynamics parameter for usage of DPP4 inhibitors. Here, we report details for the usage of a commercially available kit to evaluate DPP-enzymatic activity in vitro; and provide details on the generation of biological samples tested. Furthermore, we describe how this method can be used for evaluation of DPP activity in vivo.
Materials and Reagents
- 96 well plates, U bottom (Fisher Scientific, catalog number: 12-565-500 )
- Luminometer 96 well plates (Promega, catalog number: Z3291 )
- 2 ml Eppendorf tube (Fisher Scientific, catalog number: 05-402-7 )
- 1 ml syringe (BD, catalog number: 309659 )
- 20 G (0.9 x 25 mm) needle (BD, catalog number: 305175 )
- Luciferase-expressing transgenic mouse such as the FVB-Tg(CAG-luc,-GFP)L2G85Chco/J mouse (THE JACKSON LABORATORIES, catalog number: 008450 )
- Biologic samples such as mouse plasma/serum or organ homogenates/supernatants (see Notes)
- DPPIV-GloTM Protease Assay (Promega, catalog number: G8351 )
- 1x PBS (Thermo Fisher Scientific, GibcoTM catalog number: 14190144 )
- Recombinant human DPP4 (R&D Systems, catalog number: 1180-SE-010 )
- Prionex (Sigma-Aldrich, catalog number: G0411 )
- DPP4 inhibitor (such as K579, Sigma-Aldrich, catalog number: D3572 )
- Ethanol
Equipment
- Luminometer, with plate reader (such as Promega, model: GloMax® 96 Microplate Luminometer )
- Multichannel pipet
- XENOGEN (PerkinElmer, model: IVIS system )
- Precision scale (such as Veritas Analytical Balance, AFFORDABLE SCALES AND BALANCES, model: M124A )
- Sterile stainless steel bead 5 mm (QIAGEN, catalog number: 69989 )
- TissueLyser II (QIAGEN, catalog number: 85300 )
- Tabletop microcentrifuge (such as Eppendorf, model: 5417 R )
Procedure
文章信息
版权信息
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Barreira da Silva, R., Ingersoll, M. A. and Albert, M. L. (2017). Measurement of Dipeptidylpeptidase Activity in vitro and in vivo. Bio-protocol 7(6): e2184. DOI: 10.21769/BioProtoc.2184.
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分类
免疫学 > 免疫细胞功能 > 综合
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