Modern tools for identification of nucleic acid-binding proteins

Nadia Hégarat^{a, b}, Jean-Christophe François^{a, b,} and Danièle Praseuth^{a, b,}

^aINSERM, U565 Case Postale 26, 57 rue Cuvier, 75231 Paris Cedex 05, France

^bMuséum National d'Histoire Naturelle (MNHN) USM 503, CNRS UMR 5153 “Acides Nucleiques: dynamique, ciblage et fonctions biologiques”, Case Postale 26, 57 rue Cuvier, 75231 Paris Cedex 05, France

Available online 12 April 2008.

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Abstract

Numerous biological mechanisms depend on nucleic acid–protein interactions. The first step to the understanding of these mechanisms is to identify interacting molecules. Knowing one partner, the identification of other associated molecular species can be carried out using affinity-based purification procedures. When the nucleic acid-binding protein is known, the nucleic acid can be isolated and identified by sensitive techniques such as polymerase chain reaction followed by DNA sequencing or hybridization on chips. The reverse identification procedure is less straightforward in part because interesting nucleic acid-binding proteins are generally of low abundance and there are no methods to amplify amino acid sequences. In this article, we will review the strategies that have been developed to identify nucleic acid-binding proteins. We will focus on methods permitting the identification of these proteins without a priori knowledge of protein candidates.

Keywords: Nucleic acid-binding protein; Protein library; Affinity chromatography; Multiprotein complexes

Article Outline

1. Introduction

2. From protein libraries

3. From protein extracts

4. Identification of multiprotein complexes interacting with nucleic acids

5. Conclusions

Acknowledgements

References

Fig. 1. Protein production and immobilization on a chip. (A) A plasmid coding for a tagged protein is immobilized on an array. The microarray is incubated with rabbit reticulocyte lysate to permit fusion protein expression. Tagged protein is immobilized on chip via its interaction with an antibody directed against the tag. (B) Each ORF is cloned in a vector that is suitable for protein expression in the chosen host cell. Proteins are then purified and spotted on slides. In both cases, protein microarrays are incubated with a nucleic acid, labelled with a fluorochrome, for example. After several washes, the interacting proteins are detected using fluorescence. Using the ORF sequence, the identity of the protein is determined.

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https:/.../index.cfm?fuseaction=iProtocol.unitSectionTree&treeNodeId=9E662A36DC81D6EAF62978AC355691F1

Two-dimensional gel electrophoresis of proteins
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npg.nature.com/nmeth/journal/v2/n1/pdf/nmeth0105-83.p

TWO-DIMENSIONAL GEL ELECTROPHORESIS FOR PROTEOMICS
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proteomics.missouri.edu/aboutus/TechTips.doc

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protocols to resolve proteins based on conventional carrier ampholytes. ..... DNA sequence data: usefulness of two-dimensional gel electrophoresis and ...
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Two-dimensional fluorescence difference gel electrophoresis for ...
The methodological advance in two-dimensional gel electrophoresis (2DGE) has been the ..... TROUBLESHOOTING. Troubleshooting advice can be found in Table 1. ...
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DNA Agarose Gel Electrophoresis

Agarose Gel Electrophoresis
QUIKView DNA Stain. 25 ml WARDS QUIKView DNA Stain. 475 ml warm water (50-55° C). Agarose Gel Electrophoresis Protocol. Preparing the agarose gel ...
troi.cc.rochester.edu/~wolb/FIBR/Workshop%20Material/Gel%20Electrophoresis%20Lab%202006.doc

Agarose gel electrophoresis
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wheat.pw.usda.gov/~lazo/methods/lazo/agarose.html

Agarose Gel Electrophoresis Protocol - Biocompare
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Smeared DNA Agarose Gel Electrophoresis Troubleshooting
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Laboratory Manual for Biomedical Research

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Molecular Separation
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RNA Agarose Gel Electrophoresis
SDS-Page - Polyacrylamide gel electrophoresis
2D Page-Two - Dimensional Polyacrylamide Gel Electrophoresis
Ion Exchange Chromatography
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Gene knockout protocol
SiRNA gene knockout
Plasmid and its usefulness
DNA library construction
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Basic knowledge of microarray.
Introduction to Microarray.
MicroArray Procedure
Total RNA Isolation from cultured cells.
DNase Treatment of Total RNA
Making the single strand cDNA probe.
Automated Slide Processor (ASP) Version for hybridization.
Washing microarrays in ASP.
Processing of Array slide
Pre-hybridization of the processed slides (NON-Automated version).
Hybridization of Cy3 + Cy5 probe to glass array (NON-Automated version).
Preparation of Dendrimer Cy3 and Cy5.
Washing unbound probe from glass array (NON-Automated version).
Hybridization of Dendrimers (Cy3 and Cy5) to Array (NON-Automated version).
Washing unbound dendrimer from glass array(NON-Automated version).
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Protocol 1: Western Blot
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Genes and diseases

Genes and Disease

Most diseases are related in some way to our genes. The information contained in our genes is so critical that simple changes can lead to a severe inherited disease, make us more inclined to develop a chronic disease, or make us more vulnerable to an infectious disease...
http://www.genetics.gsk.com/link.htm