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Anal Biochem. 2015 Jul 15;481:33-42. doi: 10.1016/j.ab.2015.04.016. Epub 2015 Apr 20.

Agarose gel shift assay reveals that calreticulin favors substrates with a quaternary structure in solution.

Author information

1
Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense, Denmark; Department of Autoimmunology and Biomarkers, Statens Serum Institut, DK-2300 Copenhagen, Denmark.
2
Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense, Denmark. Electronic address: php@bmb.sdu.dk.

Abstract

Here we present an agarose gel shift assay that, in contrast to other electrophoresis approaches, is loaded in the center of the gel. This allows proteins to migrate in either direction according to their isoelectric points. Therefore, the presented assay enables a direct visualization, separation, and prefractionation of protein interactions in solution independent of isoelectric point. We demonstrate that this assay is compatible with immunochemical methods and mass spectrometry. The assay was used to investigate interactions with several potential substrates for calreticulin, a chaperone that is involved in different biological aspects through interaction with other proteins. The current analytical assays used to investigate these interactions are mainly spectroscopic aggregation assays or solid phase assays that do not provide a direct visualization of the stable protein complex but rather provide an indirect measure of interactions. Therefore, no interaction studies between calreticulin and substrates in solution have been investigated previously. The results presented here indicate that calreticulin has a preference for substrates with a quaternary structure and primarily β-sheets in their secondary structure. It is also demonstrated that the agarose gel shift assay is useful in the study of other protein interactions and can be used as an alternative method to native polyacrylamide gel electrophoresis.

KEYWORDS:

Agarose gel shift assay; Calreticulin; Direct visualization; Protein interactions

PMID:
25908558
DOI:
10.1016/j.ab.2015.04.016
[Indexed for MEDLINE]

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