Polypeptides ammonium buffers

When chromatographic resolution of species based on modifications located at the protein surface is desired, it may be advisable to use conditions that favor retention of native conformation.17 Here, the standard acidic conditions described in the preceding text may be inappropriate, and mobile phases buffered near neutrality may be required. Buffers based on ammonium acetate, ammonium bicarbonate, and triethylammonium phosphate may prove more useful in resolving polypeptide variants with differing posttranslational modifications, amino acid substitutions, or oxidation and deamidation products. The addition of more hydro-phobic ion-pairing agents may be needed to obtain polypeptide retention, and a variety of alkyl sulfonates and alkyl amines have been described for specific applications.17... 

Well-conditioned RPC can separate proteins that differ in rather small structural elements. The vasopressin-related neurophysin II, a polypeptide of 95 residues, could be separated into two isoforms that differ from each other only by one residue, either lie or Val, at position 89. This was possible by RPC on C 8 or C 18 columns (250 x 4.6 or 150 x 4.6 mm, respectively) with rather shallow gradients (6% B in 30 min or 20% B in 60 min). The gradient components were (triethyl ammonium phosphate buffer, pH 3)/acetonitrile76). 

After addition of solid ammonium sulfate(0.13g/ml) to the blue-green supernatant, precipitates formed were collected and then dissolved into phosphate buffer(20 mM, pH 8) containing 0.1-0.2% Triton X-100. P700-enriched particles thus prepared had a polypeptide composition similar to that of the CP-1 complexes isolated previously from the same cyanobacterium, showing two prominent bands of 60-67 kDa and three small bands of 14, 13 and 10 kDa by SDS-PAGE. 

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