Affinity Partitioning

The principle of affinity partitioning is to localize an affinity ligand in one phase to make it attract ligand-binding proteins. Since the phase-forming polymers are in each phase, either one can be used as ligand carrier. The standard system for affinity partitioning has been the one composed of dextran, PEG and water. Dextran is then used for localizing the ligand in the bottom phase while PEG can be used to concentrate the ligand to the top phase. PEG has often been chosen as ligand carrier because bulk proteins can be effectively partitioned into the dex-tran-rich lower phase by using high concentrations of polymers and a suitable salt. Thus, the target protein is extracted towards the upper phase leaving contaminating proteins in the bottom phase. PEG has two reactive groups (the terminal hydroxyl groups) which can be used as points of ligand attachment. In many cases only one ligand molecule is attached per PEG molecule. If the ligand is a large molecule (e.g. an antibody protein) several PEG chains may be attached to the one ligand molecule. Normally, only a fraction (1-10%) of the PEG in the two-phase system has to carry the ligand to reach maximal extraction efficiency. The more extreme the partitioning of a ligand-polymer is toward a phase the more effective it will be in extracting a ligand-binding protein into this phase. The partitioning of

Figure 2 Log Kofthe protein ribonuclease-A as function of its net charge, Z, in a two-phase systems containing KSCN (0,100 mM), KCl (•, 100 mM), or K2SO4 (□, 50 mM). System compositions: (A) 6.2%w/w dextran 500 and 4.4%w/w PEG 8000; (b) 9.8%w/w dextran 500 and 7.0%w/w PEG 8000. Protein concentration, 2gL~1. Temperature, 20°C. (Reprinted from Johansson G (1984) Molecular Cell Biochemistry 4: 169-180, with permission from Elsevier Science.)

Figure 2 Log Kofthe protein ribonuclease-A as function of its net charge, Z, in a two-phase systems containing KSCN (0,100 mM), KCl (•, 100 mM), or K2SO4 (□, 50 mM). System compositions: (A) 6.2%w/w dextran 500 and 4.4%w/w PEG 8000; (b) 9.8%w/w dextran 500 and 7.0%w/w PEG 8000. Protein concentration, 2gL~1. Temperature, 20°C. (Reprinted from Johansson G (1984) Molecular Cell Biochemistry 4: 169-180, with permission from Elsevier Science.)

the ligand-polymer should be in the same range as the non-derivatized polymer but it may, in some cases, be more extreme. The higher the polymer concentrations are in the system, i.e. the longer the tie-line of the system, the more extreme is the partitioning of PEG to the top phase and dextran to the bottom phase. This can be expressed by the partition coefficients of the two polymers:

CPEG.

CPEG, bottom

^dextran, top ^dextran, bottom where c is the respective polymer concentration in top or bottom phase. Table 1 shows the KPEG and Kdextran values for systems containing PEG 8000 and dextran 500. Dextran has a more extreme value of K than PEG, i.e. Kpeg < 1/Kdextran. Dextran should therefore, in principle, be a better ligand carrier than PEG. The concentration ratio for dextran is roughly the square of the ratio for PEG in the same system.

Solar Panel Basics

Solar Panel Basics

Global warming is a huge problem which will significantly affect every country in the world. Many people all over the world are trying to do whatever they can to help combat the effects of global warming. One of the ways that people can fight global warming is to reduce their dependence on non-renewable energy sources like oil and petroleum based products.

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