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

Sanne Grundvad Boelt, Gunnar Houen, Peter Højrup

Research output: Contribution to journalJournal articleResearchpeer-review

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.

Original languageEnglish
JournalAnalytical Biochemistry
Volume481
Pages (from-to)33-42
ISSN0003-2697
DOIs
Publication statusPublished - 2015

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Calreticulin
Sepharose
Assays
Gels
Substrates
Isoelectric Point
Proteins
Electrophoresis
Visualization
Mass spectrometry
Agglomeration

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title = "Agarose gel shift assay reveals that calreticulin favors substrates with a quaternary structure in solution",
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.",
author = "Boelt, {Sanne Grundvad} and Gunnar Houen and Peter H{\o}jrup",
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year = "2015",
doi = "10.1016/j.ab.2015.04.016",
language = "English",
volume = "481",
pages = "33--42",
journal = "Analytical Biochemistry",
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Agarose gel shift assay reveals that calreticulin favors substrates with a quaternary structure in solution. / Boelt, Sanne Grundvad; Houen, Gunnar; Højrup, Peter.

In: Analytical Biochemistry, Vol. 481, 2015, p. 33-42.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - Agarose gel shift assay reveals that calreticulin favors substrates with a quaternary structure in solution

AU - Boelt, Sanne Grundvad

AU - Houen, Gunnar

AU - Højrup, Peter

N1 - Copyright © 2015 Elsevier Inc. All rights reserved.

PY - 2015

Y1 - 2015

N2 - 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.

AB - 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.

U2 - 10.1016/j.ab.2015.04.016

DO - 10.1016/j.ab.2015.04.016

M3 - Journal article

C2 - 25908558

VL - 481

SP - 33

EP - 42

JO - Analytical Biochemistry

JF - Analytical Biochemistry

SN - 0003-2697

ER -