Polyethylene Glycol Precipitation

There is a range of polyethylene glycol (PEG) preparations available categorized by the molecular weight of the polymer. Protein solubility in the presence of PEG is influenced by the molecular weight of the PEG used, but viscosity will also increase with increasing size. As is often the case, a compromise is necessary and the most useful molecular weight range for immunoglobulin purification is in the region of 6000-10,000. There is some confusion over nomenclature in the preparations from different suppliers, with one renaming PEG 6000 as PEG 8000.

1. Clarify the serum by centrifuging at 3000g for 15 min at 4°C. Discard any precipitate and add 2 vol of 0.06 M sodium acetate, pH 4.0, at room temperature.

2. Cool 20% (w/v) solution of PEG on 0.05 M sodium phosphate, pH 7.4 to 4°C.

3. Stir the serum gently, slowly add an equal volume of the PEG solution, and continue stirring for 30 min.

4. Centrifuge at 3000g for 10 min at 4°C.

Table 1

Immunoglobulin Purification Using Caprylic Acid

Species

Volume (^L) Caprylic Acid Required/mL Serum

Human

Horse

Mouse

Sheep

Rabbit

Goat

70 55 40 70 80 80 70

5. Discard the supernatant, drain the pellet, and carefully wipe the inside of the tube to remove any remaining supernatant.

6. Resuspend the pellet in a suitable buffer and, if necessary, dialyze the solution against at least two changes of a suitable buffer at 4°C (see Note 11).

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