Name: a liquid phase affinity capture assay using magnetic beads to study protein-protein interaction: the poliovirus-nanobody example
Uploaded: 2012-05-29T12:00:00
Description: Watch this Scientific Journal Video about A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example at JoVE.com

The authors thank the staff of the department of Pharmaceutical Biotechnology and Molecular Biology and especially Monique De Pelsmacker for the preparation of the radioactive labeled virus. We are grateful to Ellen Merckx and Hadewych Halewyck for their interesting remarks and discussions and to Gerrit De Bleeser for his help in the lab. This work was financially supported by an OZR grant of the Vrije Universiteit Brussel (OZR1807), the Fonds voor Wetenschappelijk Onderzoek Vlaanderen (G.0168.10N) and the World Health Organization (TSA 200410791). Lise Schotte is a predoctoral fellow of the Fonds voor Wetenschappelijk Onderzoek Vlaanderen (FWO).

The principle (A) and an overview of the method (B) are depicted in Figure 1.
1. Preparation of the Buffer
<ol>
	<li>Prepare Binding/Wash buffer by dissolving sodium dihydrogen phosphate (50 mM) and sodium chloride (300 mM) in water and adjust the pH to 8.0. Additionally add Tween 20 (0.01% (m/v) final concentration), methionine (2% (m/v) final concentration) and albumin (0.1% (m/v) final concentration) and adjust to the required volume.</li>
</ol>
2. P...

<ol>
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In the protocol, the radioactivity of the antigen interacting with the nanobody is defined as the loss of radiolabeled virus from the supernatant. Therefore the amount of precipitated radioactivity (=100 - a %) (bound to the magnetic beads) can be estimated in an indirect way by the radioactivity in the supernatant (= a %). On the other hand, it is also possible to measure the radioactivity of the precipitated bound fraction of antigen in a direct way by eluting the immunocomplexes from the magnetic beads with 500 mM imi...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>Dynabeads His-Tag Isolation and Pulldown </td>
 <td>Invitrogen</td>
 <td>101.03D</td>
 <td>Magnetic beads</td>
 </tr>
 <tr>
 <td>Optiphase 'HiSafe' 2</td>
 <td>Perkin Elmer</td>
 <td>1200 - 436</td>
 <td>Scintillation fluid</td>
 </tr>
 </tbody>
 </table>

a liquid phase affinity capture assay using magnetic beads to study protein-protein interaction: the poliovirus-nanobody example

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

Watch this Scientific Journal Video about A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example at JoVE.com

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

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