期刊
ANALYTICAL BIOCHEMISTRY
卷 376, 期 1, 页码 73-82出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2008.02.004
关键词
acylphosphate; PhoB; phosphate stain; phospho-Asp; phosphoprotein; two-component
资金
- Howard Hughes Medical Institute Funding Source: Medline
- NIGMS NIH HHS [R37 GM047958, R37GM047958, R37 GM047958-14] Funding Source: Medline
Recent development of the phosphate chelator, Phos-tag, together with Phos-tag pendant reagents, has provided new methods for detection of phosphorylated serine, threonine, tyrosine, and histidine residues in phosphoproteins. We have investigated the use of Phos-tag for detection and quantification of phospho-aspartate in response regulator proteins that function within two-component signaling systems. Alternative methods are especially important, because the labile nature of the acylphosphate bond in response regulator proteins has restricted the application of many traditional methods of phosphoprotein analysis. We demonstrate that Phos-tag gel stain can be used to detect phospho-Asp in response regulators and that Phos-tag acrylamide gel electrophoresis can be used to separate phosphorylated and unphosphorylated forms of response regulator proteins. The latter method, coupled to Western blot analysis, enables detection of specific phosphorylated proteins in complex mixtures such as cell lysates. Standards of phosphorylated proteins can be used to correct for hydrolysis of the labile phospho-Asp bond that invariably occurs during analysis. We have employed Phos-tag methods to characterize the phosphorylation state of the Escherichia coli response regulator PhoB both in vitro, using purified protein, and in vivo, by analyzing lysates of cells grown under different conditions of induction of the PhoR/PhoB phosphate assimilation pathway. (c) 2008 Elsevier Inc. All rights reserved.
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