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

Ozden Tacal; Inci Ozer

doi:10.1016/S0165-022X(02)00070-2

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

Ozden Tacal
, Inci Ozer

Division of Biochemistry (Pharmacy)

Hacettepe University

Research output: Contribution to journal › Article › peer-review

2 Citations (Scopus)

Abstract

Horseradish peroxidase (HRP) was conjugated with bovine serum albumin (BSA) or human α₁-proteinase inhibitor (α₁-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 α₁-PI. The conjugation products were analysed both by SDS-PAGE and size exclusion chromatography (SEC) on Sephadex G200. 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.

Original language	English
Pages (from-to)	161-168
Number of pages	8
Journal	Journal of Biochemical and Biophysical Methods
Volume	52
Issue number	3
DOIs	https://doi.org/10.1016/S0165-022X(02)00070-2
Publication status	Published - Aug 2002

Keywords

Product analysis
Protein conjugation
SMCC

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10.1016/S0165-022X(02)00070-2

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

abstract = "Horseradish peroxidase (HRP) was conjugated with bovine serum albumin (BSA) or human α1-proteinase inhibitor (α1-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 α1-PI. The conjugation products were analysed both by SDS-PAGE and size exclusion chromatography (SEC) on Sephadex G200. 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.",

keywords = "Product analysis, Protein conjugation, SMCC",

author = "Ozden Tacal and Inci Ozer",

year = "2002",

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doi = "10.1016/S0165-022X(02)00070-2",

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T1 - A comparison between SDS-PAGE and size exclusion chromatography as analytical methods for determining product composition in protein conjugation reactions

AU - Tacal, Ozden

AU - Ozer, Inci

PY - 2002/8

Y1 - 2002/8

N2 - Horseradish peroxidase (HRP) was conjugated with bovine serum albumin (BSA) or human α1-proteinase inhibitor (α1-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 α1-PI. The conjugation products were analysed both by SDS-PAGE and size exclusion chromatography (SEC) on Sephadex G200. 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.

AB - Horseradish peroxidase (HRP) was conjugated with bovine serum albumin (BSA) or human α1-proteinase inhibitor (α1-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 α1-PI. The conjugation products were analysed both by SDS-PAGE and size exclusion chromatography (SEC) on Sephadex G200. 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.

KW - Product analysis

KW - Protein conjugation

KW - SMCC

UR - https://www.scopus.com/pages/publications/0036697249

U2 - 10.1016/S0165-022X(02)00070-2

DO - 10.1016/S0165-022X(02)00070-2

M3 - Article

C2 - 12376019

AN - SCOPUS:0036697249

SN - 0165-022X

VL - 52

SP - 161

EP - 168

JO - Journal of Biochemical and Biophysical Methods

JF - Journal of Biochemical and Biophysical Methods

IS - 3

ER -

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

Abstract

Keywords

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