Materials

2.1. Polymers

A variety of polymers have been used to prepare 2-phase systems (see Table 2). PEG, a linear synthetic polymer of ethyl-ene oxide units, and Dx, poly(a-1,6-glu-cose), are the most commonly used polymers in the preparation of aqueous 2-phase systems. Examples of other sugar polymers used are Ficoll (polysucrose), pullulan, and maltodextrins. Derivatized carbohydrate polymers have also been used; these include methylcellulose, hydroxyethylcel-lulose (HEC), Reppal PES (hydroxypropyl starch), benzoyl, dextran sulfate, and diethylaminoethyl (DEAE)/Dx. Examples of synthetic polymers, besides PEG, are polyvinyl alcohol (PVA), polyvinylpyrroli-done, pluronic, and random copolymers of ethylene oxide and propylene oxide: EO50PO50, EO20PO80 (UCON), etc. Several suppliers can be used worldwide: Sigma (St. Louis, MO, USA) (PEG, Dx, Dx-SO4, Ficoll, Methylcellulose), Union Carbide (Bound Brook, NJ, USA) (PEG, UCON), Amersham Pharmacia Biotech (Piscataway, NJ, USA) (Dx and Ficoll), Polysciences (Warrington, PA, USA) (PEG), Shearwater Polymers (Huntsville, AL, USA) (PEG and PEG derivatives), etc. For most applications, the polymers are used as received. Multivalent ions in commercial Dx can be eliminated by dialysis, ultrafiltration, or by a desalting step. Impurities in PEG (antioxidants, ethylene gly-col, and diethylene glycol) can be eliminated by ether or hexane precipitation of a PEG/acetone solution (1,11). Often, the molecular weight and the molecular weight distribution are given by the manufacturer. In the absence of accurate information, the molecular weight can be determined by a size exclusion chromatography-low angle light scattering tandem (no internal standards are needed) or by size exclusion chro-

matography using the appropriate standards. Some companies, like Wyatt Technology (Santa Barbara, CA, USA), provide determination of molecular weights for a fee. The molecular weights of PEG and Dx can be determined by using a Superose™ 12 column (Amersham Pharmacia Biotech) eluted with a 3% NaCl solution at room temperature (17). Figure 3 shows a typical molecular weight distribution curve for Dx. Molecular weight standards for PEG can be bought from Polysciences. Since molecular weight standards for Dx are difficult to obtain, narrow fractions of pullulan (Polysciences) can be used. Polydisperse polymers of good quality can be purchased from speciality chemical companies like Sigma. Polymer batches of narrow molecular weight distributions can also be purchased but at a much higher price. For example, less polydisperse Dx can be bought from Amersham Pharmacia

Biotech, whereas narrow fractions of PEG can be purchased from PolySciences.

2.2. Buffers

A variety of buffers have been used to regulate the pH in aqueous 2-phase systems. The two most commonly used are phosphate and Tris buffers. The buffers must be kept in a refrigerator and used within 30 days.

2.3. Additives

A series of additives are normally used in aqueous 2-phase systems. Bacteriocides (either sodium azide or chloroacetamide) are conveniently added to the polymer stock solutions or in solid form to the 2-phase system. A series of salts (shown in Figure 2) are commonly used to drive the protein of interest into one or another phase.

Figure 3. Molecular weight distribution of Dx T-500. This was obtained by running a Dx solution through a Superose 12 column eluted with NaCl (3%). The column was calibrated with pullulan standards.

2.4. Polymer Derivatives

Different PEG derivatives have been used in aqueous 2-phase partitioning experiments as affinity ligands. The three main kinds are PEG-dye, PEG-fatty acid, and PEG-imidazole compounds. Several dyes, including Cibacron Blue F3G-A (Ciba-Geigy, Basel, Switzerland), Procion Red HE-3B, Procion Green H-4G, and Procion Brown MX-5BR (I.C.I. Organic Division, Blackely, UK), and Remazol dyes (Hoechst, Frankfurt, Germany) can be conveniently attached to PEG-amine (from Shearwater Polymers). Some PEG-dye derivatives are available commercially (for example, Sigma commercializes PEG-red, MW 8000), but they can be easily prepared in the laboratory, as can other derivatives.

❖ Procedure 1. Preparation of PEG Derivatives

A. Preparation of PEG-Red

1. Dissolve aminated PEG and Procion Red HE 3B (1:1.8 ratio) in water.

2. Adjust the pH to 11.0 (5 M NaOH) and incubate the mixture at 60°C for 24 hours with constant stirring.

3. Remove the excess salts by dialysis.

B. Preparation of Iminodiacetate PEG (for Metal Affinity Two-Phase Partitioning)

1. The synthesis begins with PEG-chloride (either produced in the laboratory or obtained from Shearwater Polymers), to which iminodiacetic acid and potassium carbonate are added.

2. The solution is refluxed for 48 hours.

3. The reaction is stopped by adding sodium sulfate and allowed to separate into 2 phases.

4. The PEG-rich phase is diluted and dia-

lyzed first against sodium bicarbonate and then against water.

PEG-fatty acids can be also easily produced in the laboratory by reacting PEG with the chloride or anhydride of the fatty acid in toluene

0 0

Post a comment