A comparison between SDS-PAGE and size exclusion chromatography as analytical methods for determining product composition in protein conjugation reactions

Tacal O., Özer İ.

JOURNAL OF BIOCHEMICAL AND BIOPHYSICAL METHODS, vol.52, no.3, pp.161-168, 2002 (SCI-Expanded) identifier identifier


Horseradish peroxidase (HRP) as conjugated with bovine serum albumin (BSA) or human a,proteinase inhibitor (alpha(1)-PI). The enzyme was maleimidylated using N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and then allowed to react with thiolated BSA or reduced alpha(1)-PI. The conjugation products were analysed both by SDS-PAGE and size exclusion chromatography (SEC) on Sephadex 6200. The two methods of evaluating conjugative processes were compared with respect to information provided in relation to the behaviour of the products in solution. The results showed that neither SDS-PAGE nor SEC alone provides sufficient information about conjugate structure. The basic conjugate units observed in electrophoresis tend to form dimeric or higher-order aggregates under gel chromatographic conditions. (C) 2002 Elsevier Science B.V. All rights reserved.