एक तरल चरण आत्मीयता कब्जा चुंबकीय मोती का उपयोग करने के लिए प्रोटीन से प्रोटीन सहभागिता अध्ययन परख: उदाहरण पोलियो वायरस Nanobody
Summary
इस अनुच्छेद में, एक सरल, मात्रात्मक, तरल चरण आत्मीयता कब्जा परख प्रस्तुत किया है. यह एक विश्वसनीय तकनीक चुंबकीय मोती और एक हाथ और टैग और अन्य पर एक दूसरे, लेबल प्रोटीन (जैसे पोलियो वायरस) प्रोटीन के बीच संबंध पर टैग प्रोटीन (nanobodies जैसे) के बीच बातचीत पर आधारित है.
Abstract
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 Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred.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.
Protocol
Discussion
प्रोटोकॉल में, Nanobody के साथ बातचीत प्रतिजन की रेडियोधर्मिता radiolabeled वायरस की सतह पर तैरनेवाला से नुकसान के रूप में परिभाषित किया गया है. इसलिए उपजी रेडियोधर्मिता (= 100%) की राशि (चुंबकीय मोतियों के लिए बाध्य) स?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
लेखक रेडियोधर्मी लेबल वायरस की तैयारी के लिए औषधि जैव प्रौद्योगिकी और आण्विक जीवविज्ञान विभाग के कर्मचारियों और विशेष रूप से Monique डी Pelsmacker धन्यवाद. हम के एलेन MERCKX और उनके दिलचस्प टिप्पणी और विचार विमर्श के लिए Hadewych Halewyck और Gerrit डी Bleeser प्रयोगशाला में उसकी मदद के लिए आभारी हैं. यह काम आर्थिक रूप से Vrije Universiteit Brussel (OZR1807), Fonds वूर Wetenschappelijk Onderzoek से Vlaanderen (G.0168.10N) और विश्व स्वास्थ्य संगठन (200,410,791 टीएसए) का एक OZR अनुदान द्वारा समर्थित किया गया. Lise SCHOTTE Fonds वूर Wetenschappelijk Onderzoek Vlaanderen (FWO) के predoctoral साथी है.
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| Dynabeads His-Tag Isolation and Pulldown | Invitrogen | 101.03D | Magnetic beads |
| Optiphase ‘HiSafe’ 2 | Perkin Elmer | 1200 – 436 | Scintillation fluid |
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