Roberto Z Guzman and Javier E Garcia 1 Introduction
The partitioning in aqueous two-phase systems ATPS has become an established and well-known method for the separation and purification of biological materials 1-3 . In order to increase the selectivity and effectiveness of the separation, a ligand that partitions favorably into one of the phases and with affinity for the material of interest is introduced into the system. Thus, once binding occurs, the desired material preferentially distributes into the polymer-ligand rich phase. In general,...
Separation of Metal Ions Using an ABEC Column
1. Weigh out ca. 0.5 g of the ABEC resin prepared as outlined in Subheading 2.2.1., item 1. 2. Suspend the resin in ca. 30 mL of deionized water in a 50-mL beaker. 3. Sonicate the sample for 20 min to break up any clumps to facilitate packing the column. 4. Slurry pack the suspended resin into a small glass chromatography column. 0.5 g of resin will yield a bed height of about 2 cm in a column of the currently suggested dimensions. 5. Place a small filter frit 0.45- m pore size on top of the...
Whole Broth Extraction
1. Solubilization experiments are carried out with cell-containing cultivation medium of different cell concentrations 0.5, 1.0, 2.0, 5.0, and 10.0 w w . The different cell concentrations are achieved by harvesting and resuspension of the cells in the clarified culture medium. 2. The concentrated detergent solution is added to 10-mL samples of cell suspension to concentrations of 1.0, 2.0, 4.0, 5.0, 8.0, and 10.0 w w , in tapered graduated tubes. 3. The cell-detergent solutions are incubated...
Materials Vrk
1. Animal Tissue A wide range of animal tissue has been traditionally used for extraction of enzymes and other animal proteins. Such tissue is usually obtained from an appropriate slaughterhouse or butcher. Prices can range from a few dollars a kilo for larger tissues such as liver , to more than 1000 a kilo for small tissues that are difficult to collect, such as pituitary glands. Tissue must be as fresh as possible. see Note 1 2. Phase Forming Material Polyethylene Glycol PEG Most phase...
Equipment
The liquid-liquid extraction equipment may be classified, according to the construction and operational characteristics into two groups stagewise contactors and differential contactors. In the former type of contactors there are discrete number of stages in which two phases are equilibrated and then separated either by gravity mixer-settler and columns or by disk stack centrifuges. In the differential contactors the composition of phases changes continuously 29 , and two types can be considered...
Synthesis of Fatty Ester of PEG
Synthesis of PEG-palmitate is described here see Note 1 . 1. Dissolve 100 g of PEG in 600 mL toluene. 2. Remove moisture by azeotropic distillation by which 100 mL of toluene is removed. 3. Add trimethylamine 5 mL for PEG 3400 or 2.5 mL for PEG 8000 . 4. Add palmitoyl chloride 30 mmol for PEG 3400 or 15 mmol for PEG 8000 dropwise under stirring. 5. Reflux the mixture for 30 min. 6. Remove the salt formed by filtration. 7. Add 500 mL toluene to the filtrate and the PEG-palmitate formed is...
Torsten Minuth 1 Introduction
Extraction systems based on nonionic surfactants have been described as an alternative to the standard polymer polymer or polymer salt systems. Phase-forming surfactants are, for example, the nonionic polyoxyethylene-type detergents. This kind of aqueous two-phase system ATPS is simply induced by a switch in the temperature on the basis of the temperature-dependent reversible hydration dehydration of the polar ethylene oxide headgroups. A single isotropic micellar phase separates into two...
Virus and Polymer Properties
Table 1 gives a summary of the different animal viruses that have been subjected to two-phase extraction in aqueous polymer systems. The different genera of the virus group include a field of members, greatly varying in size and shape, as well as in surface properties. They may also differ in their requirements for stability and in adaptability to the surrounding medium. In spite of these variations among viruses, there are some polymer two-phase systems that work well with most viruses and...
Masamichi Kamihira 1 Introduction
Aqueous two-phase extraction has appreciable advantages in terms of ease of scaling-up and its applicability to systems containing solids such as cell and cell debris. However, because it is not always possible to achieve an extreme partitioning of a target molecule into either phase, the introduction of an affinity ligand to the system has been examined as a means of enhancing the specificity of partitioning. Covalent binding of a ligand to a phase-constructing polymer 1-4 see also Chapters...
Introduction Igo
Literature related to the partitioning of biological materials in aqueous polymer two-phase systems encompasses over two thousand articles and almost half a century of experience 1 . Much of this effort has involved eukaryotic cells or other biological particles. Over the past 15 years the methods and results related to these articles have been summarized in a number of books dedicated to partitioning 2-6 , as well as in various book chapters, such as ref. 7. In addition, papers from the last...
Vithaldas P Shanbhag and Poul Erik H Jensen 1 Introduction
Many biological processes, such as regulation, catalysis, and complex formation, depend on the capacity of biopolymers to interact with apolar groups, i.e., their surface or effective hydrophobicity 1 . The determination of surface hydrophobicity is therefore important for understanding the mechanism of interaction of a biopolymer with its ligands. The determination of surface hydrophobicity is also important for designing a method for the purification or the concentration of biopolymers,...
Notes Eyu
1. To date, Triton X-114 surfactant systems are widely used for protein extraction, because this surfactant is commercially available, possesses a cloud-point below 30 C and effectively solubilizes membrane-bound proteins without destroying the biological, active conformation 3,25 . Nevertheless, we prefer nonionic polyoxyethylated surfactants like C12EO5 to generate detergent-based ATPSs. There are three arguments to use polyoxyethylene glycol monoethers instead of i-octylphenoxy...
Bo Mattiasson 1 Introduction
Enzymes have been shown to operate successfully under nonphysiological conditions, e.g., in water-poor media consisting of almost pure nonpolar solvents. These new reaction conditions offer interesting possibilities to expand the scope of enzyme technology. An essential issue in this context is the method by which the enzyme is contacted with the solvent phase. A range of solid phase immobilization techniques 1,2 are available use of enzymes in microemulsions 3,4 , and chemically modified...
References Ctx
1. Hustedt, H., Kroner, K. H., Stach, W., and Kula, M.-R. 1978 Procedure for the simultaneous large-scale isolation of Pullulanase and 1,4-a-Glucan Phosphorylase from Klesbsiella pneumoniae involving liquid-liquid separations. Biotechnol. Bioeng. 20, 1989-2005. 2. Kula, M.-R., Kroner, K. H., and Hustedt, H. 1982 Purification of enzymes by liquid-liquid extraction, in Advances in Biochemical Engineering, vol. 24 Fiechter, A., ed. , Springer-Verlag, Berlin, pp. 73-118. 3. Kroner, K. H., Stach,...
Notes 1
1. Polymers PEG, a linear synthetic polymer of ethylene oxide units, and Dx, poly a-1,6-glucose , are the most commonly used polymers in the preparation of aqueous two-phase systems. Examples of other sugar polymers used are Ficoll polysucrose , pullulan and maltodextrins besides use of some derivatized carbohydrate polymers has also been reported including methylcellulose, hydroxyethylcellulose HEC , Reppal PES hydroxypropyl starch , benzoyl-dextran, dextran sulfate, and diethylaminoethyl DEAE...
The Separation of Metal Ions by ABEC
1. EIChroM Iodine Resin EIChroM Industries, Darien, IL In order to condition the ABEC resin, suspend about 3 g in deionized water and pour into a filter funnel containing 42.5 mm diam. Whatman No. 2 qualitative filter paper, to which vacuum ca. 5 in. Hg or 103 Pa is applied. The resin is washed three times in the funnel with fresh deionized water with the vacuum applied. In order to avoid excessively dehydrating the resin, the apparatus is arranged so that the air that is pulled through the...
References Aem
1. Stanier, R. Y. and Cohen-Bazire, G. 1977 Phototropic prokaryotes The cyanobacteria. Ann. Rev. Microbiol. 31, 225-274. 2. Omata, T. and Murata, N. 1984 Isolation and characterization of three types of membranes from the cyanobacterium blue-green alga Synechocystis PCC 6714. Arch. Microbiol. 139, 113-116. 3. Murata, N. and Omata, T. 1988 Isolation of cyanobacterial plasma membranes. Methods Enzymol. 167, 245-251. 4. Peschek, G., Molitor, V., Tranka, M., Wastyn, M., and Erber, W. 1988...
Preparation of WGADextran
1. Dextran T500 Amersham Pharmacia Biotech, Uppsala, Sweden is dissolved in water and freeze-dried before use. This makes it readily soluble and more convenient to use than Dextran obtained directly from the manufacturer, which dissolves very slowly. Freeze-dried Dextran retains its solubility for several months when stored dry. 2. Dichloromethane Merck, Darmstadt, Germany Dried with molecular sieves KEBO Lab, Stockholm, Sweden prior to use. 3. Dimethyl sulfoxide DMSO Merck Dried as item 2. 4....
References Yox
1. Kato, A. and Nakai, S. 1980 Hydrophobicity determined by fluorescence probe method and its correlation with surface properties of proteins. Biochim. Biophys. Acta 624, 13-20. 2. Shanbhag, V. P. and Axelsson, C.-G. 1975 Hydrophobic interaction by partition in two-phase systems. I. Partition of proteins in systems containing fatty acid esters of poly ethylene glycol . Eur. J. Biochem. 60, 17-22. 3. Axelsson, C.-G. and Shanbhag, V. P. 1976 Histone-hydrocarbon interaction. Partition of histones...
References
1. Albertsson, P. A. 1986 Partition of Cell Particles and Macromolecules, 3rd ed. Wiley, New York. 2. Flory, P. J. 1953 Principles of Polymer Chemistry. Cornell University Press, Ithaca, NY. 3. Walter, H., Brooks, D. E., and Fisher, D. eds. 1985 Partition in Aqueous Two-Phase Systems. Theory, Methods, Uses and Applications to Biotechnology. Academic, Orlando, FL. 4. Baskir, J. N., Hatton, T. A., and Suter, U. W. 1989 Protein partitioning in two-phase aqueous polymer systems. Biotechnol. Bioeng....