PubMed · 13726462
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D A ELLIS. 1961-09-09. A new universal buffer system.. https://doi.org/10.1038/1911099a0
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This work describes further improvements of coating fused silica capillaries with 2-hydroxyethyl methacrylate (HEMA) by atom transfer radical polymerization (ATRP). First, endcapping with a sterically less bulky silanyl reagent reduces the electrosmotic flow (EOF) by 25% in addition to the 40% EOF reduction caused by HEMA coating compared to a bare fused silica capillary. An additional hydrolysis step was introduced into the preparation of HEMA coated capillaries and leads to better reproducible migration times. The influence of the solvent during ATRP and the resulting polymer coating was investigated by replacement of DMF with water or water-methanol mixtures. The quality of the optimized coating was characterized by protein separations at pH 3. HEMA coated capillaries reveal up to 746000 plates. The polyvinyl alcohol (PVA) coated capillary provides only half of this efficiency. A long-term test at pH 9 shows good stability of the HEMA coated capillaries in basic medium. Also the numbers of plates in this medium was about 30% higher than for separations with the PVA capillary. In addition, the phosphate buffer was replaced by a volatile ammonium acetate buffer for later use with mass spectrometry (MS).
FT-IR spectroscopy was used to study the effects of various chaotropic and kosmotropic cosolvents (glycerol, sucrose, sorbitol, K(2)SO(4), CaCl(2), and urea) on the secondary structure and thermodynamic properties upon unfolding and denaturation of staphylococcal nuclease (Snase). The data show that the different cosolvents have a profound effect on the denaturation pressure and the Gibbs free energy (DeltaG(o)) and volume (DeltaV(o) change of unfolding. Moreover, by analysis of the amide I' infrared bands, conformational changes of the protein upon unfolding in the different cosolvents have been determined. An increase, a reduction, or an independence of the volume change of unfolding is observed, depending on the type of cosolvent, which can at least in part be attributed to the formation of a different unfolded state structure of the protein. The data are compared with the corresponding thermodynamic values of DeltaV(o) for the temperature-induced unfolding process of Snase as obtained by pressure perturbation calorimetry, and significant differences are observed and discussed.
In Saccharomyces cerevisiae, the nuclear-encoded protein Cbp1 promotes stability and translation of mitochondrial cytochrome b transcripts through interaction with the 5' untranslated region. Fusion of a biotin binding peptide tag to the C terminus of Cbp1 has now allowed detection in mitochondrial extracts by using peroxidase-coupled avidin. Cbp1 is associated with the mitochondrial membranes when high ionic strength extraction conditions are used. However, the protein is easily solubilized by omitting salt from the extraction buffer, which suggests Cbp1 is loosely associated with the membrane through weak hydrophobic interactions. Gel filtration analysis and blue native PAGE showed that Cbp1 is part of a single 900,000-Da complex. The complex was purified using the biotin tag and a sequence-specific protease cleavage site. In addition to Cbp1, the complex contains several polypeptides of molecular weights between 113 and 40 kDa. Among these, we identified another message-specific factor, Pet309, which promotes the stability and translation of mitochondrial cytochrome oxidase subunit I mRNA. A hypothesis is presented in which the Cbp1-Pet309 complex contains several message-specific RNA binding proteins and links transcription to translation of the mRNAs at the membrane.