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 nanobodies(1). Since poliovirus is sensitive to conformational conversion(2) when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred.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 polioresearch(3,4) is the micro protein A-immunoprecipitation test(5). Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies(6,7). However, as another opportunity, these interesting and stable single-domain antibodies(8) 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 (35)S 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.
Department of Pharmaceutical Biotechnology and Molecular Biology, Centre for Pharmaceutical Research, Vrije Universiteit Brussel, Belgium.
Lise
L
Schotte
Bart
B
Rombaut
Bert
B
Thys
D000906:Antibodies / Q000737:chemistry* / Q000276:immunology; D000918:Antibody Specificity; D000956:Antigens, Viral / Q000737:chemistry / Q000276:immunology; D003035:Cobalt / Q000737:chemistry; D000939:Epitopes; D006639:Histidine / Q000737:chemistry; D008280:Magnetics; D009842:Oligopeptides / Q000737:chemistry; D017955:Poliovirus / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D054730:Protein Interaction Domains and Motifs; D011506:Proteins / Q000737:chemistry*; D013462:Sulfur Radioisotopes / Q000737:chemistry
D000906:Antibodies; D000956:Antigens, Viral; D000939:Epitopes; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011506:Proteins; D013462:Sulfur Radioisotopes; D003035:Cobalt; D006639:Histidine