A single-step method to concentrate and desalt proteins which is also useful for determination of detergent binding to membrane proteins
✍ Scribed by Emilio Rivas; Nicole Pasdeloup; Marc le Maire
- Publisher
- Elsevier Science
- Year
- 1982
- Tongue
- English
- Weight
- 568 KB
- Volume
- 123
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
The conditions for obtaining highly concentrated salt-free protein solutions have been examined. It was found that a convenient and rapid method consists of combining ion-exchange and gel-filtration chromatography in one column. We observed that a volume ratio of Sephadex G-25 to ion exchanger of 2.5 is a good compromise to obtain a good recovery (97%) of a concentrated protein solution (210 mg/ml) free of salt. The procedure is also useful and accurate for measuring the detergent-to-membrane-protein-binding ratio and for preparing solutions for scattering studies.
' Abbreviations used: HA, hydroxyapatite; DDAO, dodecyl dimethylamine N-oxide; TES, N-tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid; BSA, bovine serum albumin.
MATERIALS
Chromatographic
columns of OS-, 0.9-, and 1.6-cm internal diameter were employed. Columns of OS-cm internal diameter were made with a common laboratory tube (a simple Pasteur pipet), and those of 0.9and 1.6-cm internal diameter were obtained from Pharmacia Fine Chemicals (K 9/ 15 and K 16/40). Hydroxyapatite-HT was purchased from Bio-Rad Laboratories, DEAEcellulose DE 52 from Whatman, and Sephadex G-25 medium from Pharmacia Fine Chemicals.