Sepharose protein separations

Fig. 2. Analysis of human B-receptors purified by single step immunoaflinity chromatography from T47D cytosols. B-receptors were purified by circulating cytosols from T47D cells over a column prepared by cross-linking PR-6 to protein-A-Sepharose. Proteins bound to the column were eluted with buffer at pH 11.5. Eluates were separated by SDS-PAGE. and the gels were silver stained or blotted to nitrocellulose and analyzed with PR-6.

The partition coefficient of proteins can be increased selectively by chemically attaching affinity ligands to the hydroxyl groups of the PEG. The protein can then be back-extracted from this phase by adding a fresh salt phase under conditions where the protein prefers the salt phase or by immobilizing the ligand complex in Sepharose beads, which separate into the top phase. ° Liquid-liquid extraction is commonly used in the industry because of its low cost and easy scale-up potential, but it is rarely used for protein separation even though some proteins can be effectively partitioned in ATPS. 

Gel-permeation media are extremely versatile and may be used for separation of particles such as vimses (Fig. 11) as well as proteins (34). Separations of proteins and other particles having sizes equivalent to a molecular weight of 40 x 10 are possible using the agar-based Sepharose-type gel. This particular gel has a limited temperature range for operation, however. It melts upon heating to 40°C (34). 

Lipoteichoic acids (from gram-positive bacteria) [56411-57-5J. Extracted by hot phenol/water from disrupted cells. Nucleic acids that were also extracted were removed by treatment with nucleases. Nucleic resistant acids, proteins, polysaccharides and teichoic acids were separated from lipoteichoic acids by anion-exchange chromatography on DEAE-Sephacel or by hydrophobic interaction on octyl-Sepharose [Fischer et al. Ear J Biochem 133 523 1983]. 

Separations in hydrophobic interaction chromatography have been modeled as a function of the ionic strength of the buffer and of the hydrophobicity of the column, and tested using the elution of lysozyme and ovalbumin from octyl-, butyl- and phenyl-Sepharose phases.2 The theoretical framework used preferential interaction analysis, a theory competitive to solvophobic theory. Solvophobic theory views protein-surface interaction as a two-step process. In this model, the protein appears in a cavity in the water formed above the adsorption site and then adsorbs to the phase, with the free energy change... 

Ferric ion was immobilized on a Chelating Sepharose Fast Flow column preparatory to the separation of seven enkephalin-related phosphopep-tides.17 Non-phosphorylated peptides flowed through the column, and the bound fraction contained the product. The capacity of the column was found to be 23 pmol/mL by frontal elution analysis. Cupric ion was immobilized on Chelating Superose for the isolation of bovine serum albumin.18 Cupric ion was immobilized on a Pharmacia HiTrap column for the separation of Protein C from prothrombin, a separation that was used to model the subsequent apparently successful separation of Factor IX from prothrombin Factor IX activity of the eluate was, however, not checked.19 Imidazole was used as the displacement agent to recover p-galactosidase from unclarified homogenates injected onto a nickel-loaded IMAC column.20 Pretreatment with nucleases and cleaning in place between injections were required procedures. A sixfold purification factor was observed. 

Q-Sepharose was the separation medium chosen for preparative purification of plasmid pUC18.41 Cell proteins, fragments from RNase digestion of RNA, open coil plasmid and a denatured form of the plasmid were... 

CaM was purified from porcine brain. The purity of proteins was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing. CaM and PDE were cross-linked with l-ethyl-3(3-dimethylamino-propyl) carbodiimide (EDC) or N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) in a buffer solution of 0.1 M HEPES (pH 7.1) in the presence of 1 mM CaCl2. After a buffer solution containing 2 mM EGTA added into the reaction solution, the CaM-PDE hybrid was separated from other ingredients by gel chromatography on a Sepharose CL-6B solumn. 

A detailed examination of the affinity of SLPI for the heparinized capillary was next made using a stepwise elution (from 0.1 to 0.9 M NaCl) (Fig. 11). SLPI eluted from the capillary with 0.2 M NaCl. This agreed well with results obtained by traditional affinity chromatography on a heparin-Sepharose matrix. The ACE method has the unique advantages over traditional affinity chromatography in that it requires much smaller quantities of protein and afforded better separation profiles. 

Klostermeyer (1975) used an activated thiol-Sepharose 4B column with Tris-HCl buffer containing dithiothreitol to separate the K-and aa2-caseins from the aai-and /3-caseins in whole casein. More recently, Creamer and Matheson (1981) studied the fractionation of casein by hydrophobic interaction chromatography on octyl- or phenyl-Sephar-ose CL-4B columns. The whole casein was adsorbed onto the column from dilute phosphate buffers. A gradient of 0 to 40% ethylene glycol followed by 6 M urea was employed to desorb the protein. Optimum separation was obtained with an increasing urea gradient. Under all conditions, the major /3-casein component was eluted more readily than the asi-casein in spite of its higher hydrophobicity. 

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