荧光肽酶谱法检测蛋白酶活性
Summary
在这里, 我们提出了一个详细的协议, 修改酶图技术, 其中荧光肽被用作可降解的底物, 而不是原生蛋白质。生物样品在荧光肽酶谱中的电泳使检测到比以前的酶学技术更广泛的蛋白酶。
Abstract
该方法的目的是测量复杂生物样品的蛋白溶解活性。通过嵌入可降解底物的解析凝胶, 通过电泳对样品进行分子量分离。这种方法不同于传统的凝胶酶学, 因为淬火的荧光肽是共价地纳入解决凝胶, 而不是全长蛋白质, 如明胶或酪蛋白。使用荧光肽可以直接检测蛋白溶解活性, 而无需额外的染色步骤。生物样品中的酶将淬火的荧光肽裂开, 从而增加荧光。然后用标准的荧光凝胶扫描仪对凝胶中的荧光信号进行成像, 并使用密度测量进行量化。多肽作为可降解底物的使用极大地扩展了利用酶成像技术检测到的可能的蛋白酶。
Introduction
凝胶酶学是一种生物技术, 用于测量生物样本中的蛋白溶解活性, 如体液或细胞培养介质 1,2,3。样品通过嵌入可降解基板的聚丙烯酰胺凝胶, 通过电泳的分子量分离。常见的可降解底物包括明胶、酪蛋白、胶原蛋白和弹性蛋白, 这些物质已被用来测量基质金属蛋白酶 (mmp)-1、-2、-3、-7、-8、-9 和-11, 此外还有各种导管 1, 2,4 个,5,6,7.,8. 电泳后, 酶被重新修饰, 并允许降解凝胶内的蛋白质。在传统的凝胶酶学中, 凝胶被蛋白质染料 (如 Coomassie) 染色, 蛋白酶活性被检测为信号的损失,即深蓝色背景上的白色带 (蛋白质降解)。
在这里, 我们描述了一种替代的凝胶酶学方法的协议, 其中可降解的底物是一个短的, 荧光肽共价地纳入聚丙烯酰胺凝胶 (图 1)。与传统的凝胶酶学相比, 合成肽作为可降解的底物的替代, 可以检测到更广泛的蛋白酶, 而传统的凝胶酶学方法是与原生蛋白质9相比。荧光肽的共价连接可防止凝胶电泳过程中的肽扩散和迁移, 用以前的方法9,10观察到。此外, 使用氟基板可以直接检测蛋白酶活性, 而无需额外的染色和去染色步骤。该方法的总体目标是通过在合氧化酶凝胶中共价地加入氟生成肽来检测生物样品中的蛋白酶活性。
Protocol
1. 解决凝胶层的制备 根据表 1准备10% 的聚丙烯酰胺解析凝胶溶液。在浇注凝胶之前立即加入四甲基二胺 (temed) 和过硫酸铵 (aps), 因为它们的添加会引发聚合反应。 用10% 的解析凝胶溶液填充一个空的1.5 毫米小凝胶盒 (5 毫升)。 在聚丙烯酰胺凝胶顶部添加一层薄薄的异丙醇 (~ 500μl), 以产生水平凝胶并防止气泡。使用剩余的聚丙烯酰胺溶液来跟踪聚合反应的进展。?…
Representative Results
使用此处描述的方法, 将两种荧光蛋白酶降解肽纳入聚丙烯酰胺凝胶中: ggpqg iwqk (peg) 2c(在整个文本和数字中简称 qgiw) 和 gpla cpMeOBzl wark(peg) 2c (在整个案文和数字中缩写为 lacw)。表示的部位。qgiw 是一个用于检测细胞胶原酶14的胶原蛋白-i 派生序列。lacw 是一个针对检测 mmp-14 和 mmp-1115 进行优化的序列。这些肽用 n-?…
Discussion
目前的酶学技术依赖于将原生底物纳入聚丙烯酰胺凝胶来检测蛋白溶解。虽然这些技术已经得到广泛的使用, 他们仍然是有限的蛋白酶, 他们可以检测到的数量。在这里, 描述了一个协议, 其中荧光, 蛋白酶降解肽被纳入聚丙烯酰胺解析凝胶。使用 azidi-pga-马来酰亚胺链接器分子进行共价偶联, 可分离和检测比目前本地底物更广泛的蛋白酶。荧光肽的高度可调谐特性使研究人员能够设计出能够针对其?…
Declarações
The authors have nothing to disclose.
Acknowledgements
由俄亥俄州立大学工程学院生物医学工程系和综合癌症中心—-arthur g. james 肿瘤医院和 richard j. solove 研究所提供的资金。
Materials
| 1.5 mm Empty Gel Cassettes | ThermoFisher Scientific | NC2015 | |
| 1.5 mm, 10 well Empty Gel Cassette Combs | ThermoFisher Scientific | NC3510 | |
| 1x Phosphate Buffered Saline | Fisher Scientific | 10-010-049 | |
| 20% SDS Solution | Ambion | AM9820 | |
| 3x Zymography Sample Buffer | Bio-Rad | 1610764 | |
| 40% (w/v) Acrylamide/Bis (19:1) | Ambion | AM9022 | |
| 6 Well Tissue Culture Plates | ThermoFisher Scientific | 087721B | |
| Amicon Ultra-2 Centrifugal Filter Unit (10 kDa MWCO) | Sigma-Aldrich | UFC201024 | |
| Ammounium Persulfate | Sigma-Aldrich | A3678 | |
| Azido-PEG3-Maleimide Kit | Click Chemistry Tools | AZ107 | |
| Calcium Chloride | ThermoFisher Scientific | BP510100 | |
| Dimethyl Sulfoxide | Fisher Scientific | BP231 | |
| Isopropanol | Fisher Scientific | A416P | |
| Micro BCA Protein Assay Kit | ThermoFisher Scientific | 23235 | |
| N N N' N'-Tetramethylethylenediamine (TEMED) | Sigma-Aldrich | T9281 | |
| PowerPac Basic Power Supply | Bio-Rad | 1645050 | |
| Precision Plus Protein Dual Color Standard | Bio-Rad | 161-0374 | |
| PrecisionGlide Hypodermic Needles | Fisher Scientific | 14-826 | |
| Round Bottom Flask (100 mL) | Fisher Scientific | 50-873-144 | |
| Septum Rubber Stopper | Fisher Scientific | 50-872-546 | |
| Sterile Slip Tip Syringe (1 mL) | Fisher Scientific | 14-823-434 | |
| Triton X-100 | Sigma-Aldrich | X100 | |
| Trizma hydrochlroide | Sigma-Aldrich | T5941 | |
| Typhoon 9410 Molecular Imager | GE Amersham | 8149-30-9410 | |
| Zinc Chloride | Sigma-Aldrich | 208086 |
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Citar este artigo
Deshmukh, A. A., Weist, J. L., Leight, J. L. Detection of Protease Activity by Fluorescent Peptide Zymography. J. Vis. Exp. (143), e58938, doi:10.3791/58938 (2019).