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Labeling Proteins (labeling + protein)

Distribution by Scientific Domains

Chemistry	50%

Selected Abstracts

Probing protein function by chemical modification,

JOURNAL OF PEPTIDE SCIENCE, Issue 10 2010
Yao-Wen Wu
Abstract Labeling proteins with synthetic probes, such as fluorophores, affinity tags, and other functional labels is enormously useful for characterizing protein function in vitro, in live cells, or in whole organisms. Recent advancements of chemical methods have substantially expanded the tools that are applicable to modify proteins. In this review, we discuss some important chemical methods for site-specific protein modification and highlight the application of established techniques to tackle biological questions. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd. [source]

Extravascular Transport of Fluorescently Labeled Albumins in the Rat Mesentery

MICROCIRCULATION, Issue 3 2002
NORMAN R. HARRIS
ABSTRACT Objective: Fluorescently labeled albumin is used frequently as a tracer when monitoring microvascular permeability. Several fluorescent dyes are available for labeling protein, including fluorescein isothiocyanate (FITC) and Texas Red (TR). Because differences in leakage of dye-labeled proteins have been reported, the objective of the present study was to compare the accumulation of these two tracers in interstitium and lymph after the inflammatory event of ischemiareperfusion. Methods: Anesthetized rats were injected intravenously with FITC-labeled albumin (FITC-alb) and TR-labeled albumin (TR-alb) before 30 minutes of mesenteric ischemia. Because the tracers leaked out of the microcirculation after reperfusion, accumulation in the surrounding buffer-superfused tissue, and in separate experiments, accumulation in lymph vessels, was defined as the ratio of tissue-to-plasma and lymph-to-plasma fluorescence. Results: Reperfusion induced a significant increase in tissue-to-plasma fluorescence of FITC-alb; however, no increase was observed for TR-alb. In contrast, lymph-to-plasma fluorescence of TR-alb tended to be greater than FITC-alb. Reperfusion-induced increases in tissue-to-plasma fluorescence of TR-alb occurred only when the superfusate was replaced with mineral oil, in which case tissue-to-plasma TR-alb fluorescence tended to be greater than FITC-alb. Conclusions: Measurement of fluorescently labeled albumin leakage from mesenteric venules depends on the dye used to label the albumin and requires an assessment of losses from the extravascular measuring region. [source]

Thiol-reactive dyes for fluorescence labeling of proteomic samples

ELECTROPHORESIS, Issue 14 2003
Kamala Tyagarajan
Abstract Covalent derivatization of proteins with fluorescent dyes prior to separation is increasingly used in proteomic research. This paper examines the properties of several commercially available iodoacetamide and maleimide dyes and discusses the conditions and caveats for their use in labeling of proteomic samples. The iodoacetamide dyes BODIPY TMR cadaverine IA and BODIPY Fl C1 -IA were highly specific for cysteine residues and showed little or no nonspecific labeling even at very high dye:thiol ratios. These dyes also showed minimal effects on pI's of standard proteins. Some iodoacetamide dyes, (5-TMRIA and eosin-5-iodoacetamide) and some maleimide dyes (ThioGlo I and Rhodamine Red C2 maleimide) exhibited nonspecific labeling at high dye:thiol ratios. Labeling by both iodoacetamide and maleimide dyes was inhibited by tris(2-carboxyethyl)phosphine (TCEP); interactions between TCEP and dye were also observed. Thiourea, an important component of sample solubilization cocktails, inhibited labeling of proteins with iodoacetamide dyes but not with maleimide dyes. Maleimide dyes may serve as an alternative for labeling proteins where it is essential to have thiourea in the solubilization buffer. Covalent derivatization by BODIPY TMR cadaverine IA, BODIPY Fl C1 -IA or Rhodamine Red C2 maleimide was also demonstrated to be compatible with in-gel digestion and peptide mass fingerprinting by matrix assisted laser desorption/ionization-mass spectrometry and allowed successful protein identification. [source]

Quantification of change in phosphorylation of BCR-ABL kinase and its substrates in response to Imatinib treatment in human chronic myelogenous leukemia cells

PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 16 2006
Xiquan Liang Dr.
Abstract Phosphorylation by the constitutively activated BCR-ABL tyrosine kinase is associated with the pathogenesis of the human chronic myelogenous leukemia,(CML). It is difficult to characterize kinase response to stimuli or drug treatment because regulatory phosphorylation events are largely transient changes affecting low abundance proteins. Stable isotope labeling with amino acids in cell culture,(SILAC) has emerged as a pivotal technology for quantitative proteomics. By metabolically labeling proteins with light or heavy tyrosine, we are able to quantify the change in phosphorylation of BCR-ABL kinase and its substrates in response to drug treatment in human CML cells. In this study, we observed that BCR-ABL kinase is phosphorylated at tyrosines,393 and 644, and that SH2-domain containing inositol phosphatase (SHIP)-2 and downstream of kinase (Dok)-2 are phosphorylated at tyrosine,1135 and 299, respectively. Based on the relative intensity of isotopic peptide pairs, we demonstrate that the level of phosphorylation of BCR-ABL kinase as well as SHIP-2 and Dok-2 is reduced approximately 90% upon treatment with Imatinib, a specific inhibitor of BCR-ABL kinase. Furthermore, proteins, such as SHIP-1, SH2-containing protein (SHC) and Casitas B-lineage lymphoma proto-oncogene (CBL), are also regulated by Imatinib. These results demonstrate the simplicity and utility of SILAC as a method to quantify dynamic changes in phosphorylation at specific sites in response to stimuli or drug treatment in cell culture. [source]