期刊
ANALYTICAL BIOCHEMISTRY
卷 396, 期 1, 页码 117-123出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2009.09.001
关键词
Contamination; Diffusion; DOSY; Lectin; Protein purification; Relaxation
资金
- Instituto de Tecnologia Quimica e Biologica (ITQB)
- EC [MRTN-CT-2005-019561]
- Ministry of Science of Innovation of Spain [CTQ2006-10874-C02-01, CFQ2009-8536]
- Fundacao para a Ciencia e a Tecnologia (Portugal)
- Fundo Europeo de Desenvolvimento Regional (FEDER)
Small molecules are difficult to detect in protein solutions, whether they originate from elution during affinity chromatography (e.g., imidazole, lactose), buffer exchange (Tris), stabilizers (e.g., beta-mercaptoethanol, glycerol), or excess labeling reagents (fluorescent reagents). Mass spectrometry and high-pressure liquid chromatography (HPLC) often require substantial efforts in optimization and sample manipulation to provide sufficient sensitivity and reliability for their detection. One-dimensional (1 D) H-1 nuclear magnetic resonance (NMR) Could, in principle, detect residual amounts of small molecules in protein solutions down to equimolecular concentrations with the protein. However, at lower concentrations, the NMR signals of the contaminants can be hidden in the background spectrum of the protein. As an alternative, the 1 D diffusion difference protocol used here is feasible. It even improves the detection level, picking up NMR signals from small-molecule contaminants at lower concentrations than the protein itself. We successfully observed 30 mu M imidazole in the presence of four different proteins (1-1.5 mg/ml, 6-66 kDa, 25-250 mu M) by I D diffusion-ordered spectroscopy (DOSY) difference and 1-h total acquisition time. Of note, imidazole was not detected in the corresponding 1 D H-1 NMR spectra. This protocol can be adapted to different sample preparation procedures and NMR acquisition methods with minimal manipulation in either deuterated or nondeuterated buffers. (C) 2009 Elsevier Inc. All rights reserved.
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