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The original protocol of two-dimensional electrophoresis with immobilized pH gradient (IPG-Dalt; Gorg et al., Electrophoresis 1988, 9, 531-546) is updated. Merits and limits of different methods for sample solubilization, sample application (by cup-loading or ingel rehydration) with respect to the pH interval used for IPG-isoelectric focusing are critically discussed. Guidelines for running conditions of analytical and micropreparative IPG-Dalt, using wide IPGs up to pH 12 for overview patterns, or narrow IPGs for zoom-in gels for optimum resolution and detection of minor components, are stated. Results with extended separation distances as well as automated procedures are demonstrated, and a comparison between protein detection by silver staining and fluorescent dyes is given. A brief trouble shooting guide is also included.  相似文献
2.
The rising number of proteome projects leads to new challenges for two-dimensional electrophoresis with immobilized pH gradients and different applications of this technique. Not only wide pH gradients such as 4-12 or 3-12 (Görg et al., Electrophoresis 1999, 20, 712-717) which can give an overview of the total protein expressions of cells are in demand but also overlapping narrow immobilized pH gradients are to be used for more specialized and detailed research and micropreparative separations. The advantage of overlapping narrow pH gradients is the gain in higher resolution by stretching the protein pattern in the first dimension. This simplifies computer-aided image analysis and protein identification (e.g., by mass spectrometry). In this study the protein patterns of yeast cells in pH gradients 4-5, 4.5-5.5, 5-6, 5.5-6.7 and 6-9 are presented and compared to the pH 4-7 and 3-10 gradients. This combination allowed us to reveal a total of 2286 yeast protein spots compared to 755 protein spots in the pH 3-10 gradient.  相似文献
3.
A general route to azadepsipeptides, a new class of pseudopeptides, has been established. The methodology was applied to the synthesis of a bis-aza analogue of the antiparasitic cyclooctadepsipeptide PF1022A. Comparison of the X-ray crystal structures of natural PF1022A (8) and the chimeric aza analogue 9 revealed that the introduction of nitrogen in the backbone of PF1022A results in almost complete conservation of the 3D structure with only minor deviations at the new nitrogen positions.  相似文献
4.
Addition of the Allenic amine H2C═C═CHNMe2 and the corresponding N-morpholinoallene to a solution of an aromatic or heteroaromatic aldehyde, RCH═O, and lithium bromide in tetrahydrofuran followed by treatment of the reaction mixture with a small amount of acetic acid at elevated temperatures affords the aldehydes RCH═C(CH2NR'2)CH═O (NR'2=NMe2 or morpholino) in reasonable yields.  相似文献
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