Practical Protein Chemistry - A. Darbre 1989

Determination of the composition of protein oligomers. Preparation of monomers and polypeptide chains
Preparation of monomers for sequencing
Techniques for working with lipoproteins

Apoproteins are usually prepared from Lipoproteins by extracting Lipids with organic Solvents (such as ethanol, ether–ethanol, or methanol–chloroform mixtures); however, some apoproteins are partially soluble in organic solvents. Serum lipoproteins contain a fraction of low-molecular-weight Peptides that are soluble in ethanol and, to a lesser extent, in an ethanol–ether mixture, whereas high-molecular-weight lipoproteins are insoluble in organic solvents [157]. The solubility of apoproteins in organic solvents decreases as the Temperature is lowered. Evidence also indicates that certain Membrane Proteins contain covalently linked lipids [159].

1.6.3.1. Procedures.

Extraction of low-density lipoproteins [157]. The extraction is carried out at —10 °C. A protein solution (2–5 mg/mL) is dialyzed against 0.15 M NaCl containing 1 mM EDTA, and then added dropwise to 50 mL of an ether–95% ethanol mixture (1:3) in a 50 mL round-bottom centrifuge tube. Extraction is performed for 2 h with slow Rotation of the tube (15 rpm). Finally, the precipitate is separated by centrifugation at 2000 rpm for 10 min. A second extraction is conducted in a similar manner. The precipitate is then washed three times with ether and dried under a nitrogen atmosphere. To recover any protein that has partially dissolved in the organic solvent, the first extract is diluted with ether (to an ethanol-to-ether ratio of 3:5) and kept at —10 °C for 12 h.

Extraction of chicken egg yolk lipoproteins [30]. A lipoprotein solution in 1 M NaCl (~6% w/v) is dialyzed several times against 6 M urea + 0.05 M HCl (pH 3.3) at 20 °C until NaCl is almost completely removed. To accelerate desalination, preliminary dialysis against distilled Water can be performed. A chloroform–methanol mixture (20 mL + 40 mL) is added to the resulting solution (17 mL), and the homogeneous solution is incubated at 20–25 °C for 30 min, after which an additional 20 mL of chloroform is added. The lower layer is discarded, and the upper layer is re-extracted with 10 mL of chloroform. If necessary, the emulsion is centrifuged. The chloroform layer is discarded, and the aqueous layer is kept at 20 °C for 20 h before being dialyzed against 6 M urea.