A HaloTag-based small molecule microarray screening methodology with increased sensitivity and multiplex capabilities.
A HaloTag-based small molecule microarray screening methodology with increased sensitivity and multiplex capabilities.
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DOI：
10.1021/cb300453k
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发表时间：
2012-12-21
影响因子：
4
通讯作者：
Crews, Craig M.
中科院分区：
文献类型：
--
作者：
Noblin, Devin J.;Page, Charlotte M.;Tae, Hyun Seop;Gareiss, Peter C.;Schneekloth, John S.;Crews, Craig M.
小分子微阵列（SMMs）代表了一个通用的平台，用于筛选不依赖于靶蛋白功能抑制的小分子-蛋白质相互作用。为了增加SMM的范围和实用性，我们修改了SMM筛选方法，以提高检测灵敏度并促进多重筛选。将靶蛋白融合到HaloTag蛋白上，使我们能够用荧光团共价预标记融合蛋白，从而提高检测灵敏度和进行多重筛选的能力。我们使用FKBP 12和两个配体（雷帕霉素和ARIAD的“bumpxs”配体）之间的相互作用来显示基于HaloTag的SMM筛选方法显著增加测定灵敏度。此外，使用野生型FKBP 12和FKBP 12 F36 V突变体，我们表明，用不同的荧光团预标记各种蛋白质亚型，使我们能够进行多重筛选，并确定特定亚型的配体。最后，我们表明，这种多重筛选技术是能够确定一个特定的PTP 1B亚型选择性配体使用20,000化合物筛选甲板。
Small Molecule Microarrays (SMMs) represent a general platform for screening small molecule–protein interactions independent of functional inhibition of target proteins. In an effort to increase the scope and utility of SMMs, we have modified the SMM screening methodology to increase assay sensitivity and facilitate multiplex screening. Fusing target proteins to the HaloTag protein allows us to covalently prelabel fusion proteins with fluorophores, leading to increased assay sensitivity and an ability to conduct multiplex screens. We use the interaction between FKBP12 and two ligands, rapamycin and ARIAD’s “bumpxs” ligand, to show that the HaloTag-based SMM screening methodology significantly increases assay sensitivity. Additionally, using wild type FKBP12 and the FKBP12 F36V mutant, we show that prelabeling various protein isoforms with different fluorophores allows us to conduct multiplex screens and identify ligands to a specific isoform. Finally, we show this multiplex screening technique is capable of identifying ligands selective for a specific PTP1B isoform using a 20,000 compound screening deck.
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影响因子：
15.3
作者：
You-Ten, K E;Muise, E S;Itie, A;Michaliszyn, E;Wagner, J;Jothy, S;Lapp, W S;Tremblay, M L
通讯作者：
Tremblay, M L
影响因子：
3.2
作者：
Tae, Hyun Seop;Sundberg, Thomas B.;Neklesa, Taavi K.;Noblin, Devin J.;Gustafson, Jeffrey L.;Roth, Anke G.;Raina, Kanak;Crews, Craig M.
通讯作者：
Crews, Craig M.
影响因子：
7.3
作者：
Comeau, Anthony B.;Critton, David A.;Seto, Christopher T.
通讯作者：
Seto, Christopher T.
影响因子：
--
作者：
Bradner, James E.;McPherson, Olivia M.;Koehler, Angela N.
通讯作者：
Koehler, Angela N.
影响因子：
15
作者：
MacBeath, G;Koehler, AN;Schreiber, SL
通讯作者：
Schreiber, SL