Related Experiment Video
Updated: Aug 7, 2026

Purification of Native Complexes for Structural Study Using a Tandem Affinity Tag Method
Published on: July 27, 2016
Characterization of the ERp57-Tapasin complex by rapid cellular acidification and thiol modification
Antony N Antoniou1, Simon J Powis
1Division of Cell Biology and Immunology, School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland.
Abstract:
Major histocompatibility complex (MHC) class I molecules bind and present short peptides to cells of the immune system. The oxidoreductase ERp57 is involved in the assembly of MHC class I molecules and is a component of the peptide loading complex, where it is found disulfide-bonded to tapasin. We have studied ERp57 and the ERp57-tapasin conjugate by rapid acidification of the intracellular environment with trichloroacetic acid (TCA), followed by thiol modification with the alkylating agent 4'-maleimidylstilbene-2,2'-disulfonic acid (AMS). By using TCA/AMS treatment, non-tapasin-associated ERp57 is shown to exist almost exclusively in a reduced state, suggesting that both thioredoxin-like CXXC motifs are exposed and reduced. A 110-kDa product is readily detected with this TCA/AMS protocol and is confirmed as an ERp57-tapasin conjugate by its absence from the tapasin-deficient .220 cell line and by immunoblotting with both ERp57- and tapasin-specific antisera. The ERp57-tapasin conjugate can also be modified with the oxidizing agent diamide, indicating that within the pool of ERp57-tapasin complexes the free, non-tapasin-linked CXXC motif exists in both oxidized and reduced states, suggesting availability to undergo redox reactions.
More Related Videos
Related Concept Videos
Tail-anchoring of Proteins in the ER Membrane
Protein Modifications in the RER
Broadly, these modifications can be categorized into four main categories — glycosylation, formation of disulfide bonds, assembly of protein subunits, and specific proteolytic cleavages like removal of signal sequences.
Protein Folding Quality Check in the RER
The Unfolded Protein Response
Export of Misfolded Proteins out of the ER

