Microfluidic Printing-Based Method for the Multifactorial Study of Cell-Free Protein Networks.
Microfluidic Printing-Based Method for the Multifactorial Study of Cell-Free Protein Networks.
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DOI：
10.1021/acs.analchem.2c01851
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发表时间：
2022-08-09
影响因子：
7.4
通讯作者：
Pan, Tingrui
中科院分区：
文献类型：
--
作者：
Zhou, Chuqing;Shim, Jiyoung;Fang, Zecong;Meyer, Conary;Gong, Ting;Wong, Matthew;Tan, Cheemeng;Pan, Tingrui
蛋白质网络可以在体外组装，用于基础生物化学研究，药物筛选和人工细胞的创建。使用两种标准方法：手动移液;移液机器人。手动移液在输入试剂的数量和单个样品中试剂的组合方面具有有限的通量。虽然移液机器人在提高移液效率和节省动手时间方面很明显，但它们的液体处理量通常在几微升到几百微升之间。微流控方法已被开发，以尽量减少试剂消耗和加速筛选，但在多因子蛋白质研究中具有挑战性，因为它们依赖于复杂的结构和标记染料。在这里，我们设计了一种新的基于冲击打印的方法来生成包含油包水液滴的打印微滴阵列。打印的液滴体积与单个液滴数量成线性比例（R2=0.9999），并且每个单个液滴体积为约59.2nL（变异系数=93.8%）。我们的新方法使蛋白质网络的研究在两个膜结合和结合状态，没有和锚脂DGS-NTA（Ni）分别。使用丝裂原活化蛋白激酶（MAPK）的子网络的方法证明。在每个反应位点使用< 1 μL的反应体积，制备100个不同的基于液滴的反应需要不到10分钟。我们验证了MEK 1（R4 F）* 和ERK 2 WT在不同浓度下单独和一起的激酶（ATP酶）活性，没有和有选择性膜附着。我们的新方法为蛋白质网络研究和相关应用提供了一种节省试剂、高效和灵活的方法。
Protein networks can be assembled in vitro for basic biochemistry research, drug screening, and the creation of artificial cells. Two standard methodologies are used: manual pipetting; pipetting robots. Manual pipetting has limited throughput in the number of input reagents and the combination of reagents in a single sample. While pipetting robots are evident in improving pipetting efficiency and saving hands-on time, their liquid handling volume usually ranges from a few to hundreds of microliters. Microfluidic methods have been developed to minimize the reagent consumption and speed up screening, but are challenging in multifactorial protein studies due to their reliance on complex structures and labeling dyes. Here, we engineered a new impact-printing-based methodology to generate printed microdroplet arrays containing water-in-oil droplets. The printed droplet volume was linearly proportional (R2=0.9999) to the single droplet number, and each single droplet volume was around 59.2 nL (Coefficient of Variation=93.8%). Our new methodology enables the study of protein networks in both membrane-unbound and -bound states, without and with anchor lipids DGS-NTA(Ni) respectively. The methodology is demonstrated using a sub-network of mitogen-activated protein kinase (MAPK). It takes less than 10 minutes to prepare 100 different droplet-based reactions, using < 1 μL reaction volume at each reaction site. We validate the kinase (ATPase) activity of MEK1 (R4F) * and ERK2 WT individually and together under different concentrations, without and with the selective membrane attachment. Our new methodology provides a reagent-saving, efficient and flexible way for protein network research and related applications.
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影响因子：
16
作者：
通讯作者：
--
影响因子：
7.4
作者：
Sun, Shuwen;Slaney, Thomas R.;Kennedy, Robert T.
通讯作者：
Kennedy, Robert T.
影响因子：
16.8
作者：
Hui E;Vale RD
通讯作者：
Vale RD
DOI：
10.1126/science.aaz6802
发表时间：
2020-05-08
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Miller TE;Beneyton T;Schwander T;Diehl C;Girault M;McLean R;Chotel T;Claus P;Cortina NS;Baret JC;Erb TJ
通讯作者：
Erb TJ
影响因子：
7.4
作者：
Rane, Tushar D.;Zec, Helena C.;Wang, Tza-Huei
通讯作者：
Wang, Tza-Huei