Protein aggregation mediated by cysteine oxidation during the stacking phase of discontinuous buffer SDS-PAGE.

Abstract:

:The resolution of complex protein mixtures by discontinuous buffer SDS-PAGE is accomplished by their concentration into thin bands in the stacking gel, followed by their separation during migration through the resolving gel. Recombinant human interferon-inducible protein-10 (IP-10), a 10-kDa C-X-C chemokine with four cysteines, aggregated during the stacking phase of SDS-PAGE and generated a band with an apparent molecular mass of 18 kDa. This aggregation depended on the presence of reduced sulfhydryl residues on IP-10, on the amount of loaded protein, and on the concentration of the ammonium persulfate used to polymerize the stacking gel. The aggregation of IP-10 could be prevented by reduction of its sulfhydryls with dithiothreitol followed by irreversible blockade with iodoacetamide. These methods may be useful in the prevention of aggregation of sulfhydryl-containing proteins during SDS-PAGE, especially when large quantities are analyzed to assess their purity.

journal_name

Biotechniques

journal_title

BioTechniques

authors

Crow MK,Karasavvas N,Sarris AH

doi

10.2144/01302st04

subject

Has Abstract

pub_date

2001-02-01 00:00:00

pages

311-6

issue

2

eissn

0736-6205

issn

1940-9818

journal_volume

30

pub_type

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