Practical Protein Chemistry - A. Darbre 1989

Determination of the composition of protein oligomers. Preparation of monomers and polypeptide chains
Preparation of monomers for sequencing
Desalting

Typically, monomers or polypeptide chains are desalted using dialysis or Gel filtration. Structure/19.html">The Importance of desalting should not be underestimated. Sometimes, prior to dialyzing against Water, it is advisable to dialyze the sample against dilute electrolytes or Buffer solutions. In this case, the sample will retain its solubility in water after exhaustive desalting. Special Cells are commercially available for small-volume dialysis.

Gel filtration on pre-prepared columns (Section 1.2.2.1) is a faster desalting method. The eluent and matrix parameters are chosen According to the solubility and Molecular Weight of the protein under study. Sparingly soluble hydrophobic Proteins are dissolved in 98% formic acid, which is diluted to a 75% concentration prior to Sample application on the Column (Section 1.3.2.1). Proteins with molecular weights of 5,000–30,000 are desalted on Sephadex G-25, while higher-molecular-weight proteins are desalted on Sephadex G-50. The sample volume should not exceed 1/3 to 1/4 of the column volume. Pharmacia produces Sephadex G-25 columns designed for rapid (within 5 min) desalting of 2.5 ml samples.

1.6.5.1. Performance of desalting [83]. Small Peptides are desalted on Bio-Gel P-2 columns in 1 M acetic acid or on Sephadex G-10 columns in 1 M NH4OH, followed by lyophilization.

Large Peptides and Proteins are dialyzed three times for 12 h against 0.15 M NaCl containing 0.1% SDS, and then three times against 0.02% SDS. If the protein was isolated by SDS-PAGE, the eluate is centrifuged at 20,000 rpm for 20 min to remove acrylamide gel particles. Coomassie dye impurities do not interfere with sequencing. However, it should be borne in mind that the equilibrium distribution of SDS across a dialysis membrane is a slow process and may take several days. Thus, the final SDS concentration in the sample depends on the duration of dialysis.

Electrodialysis can also be used for sample desalting (Section 17.3.1).



Last update: 06/08/2026

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