Mammalian Expression Cloning of Nucleic Acid Binding Proteins by Agarose Thin-Layer Gelshift Clone Selection
Here we report a functional screening technique to identify cDNAs encoding mammalian nucleic acid binding proteins. We have combined cDNA expression cloning with the agarose thin-layer gelshift assay technique to detect specific nucleic acid binding proteins from a mammalian expression library. We d...
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| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Taylor & Francis Group
2002-10-01
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| Series: | BioTechniques |
| Online Access: | https://www.future-science.com/doi/10.2144/02334rr03 |
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| Summary: | Here we report a functional screening technique to identify cDNAs encoding mammalian nucleic acid binding proteins. We have combined cDNA expression cloning with the agarose thin-layer gelshift assay technique to detect specific nucleic acid binding proteins from a mammalian expression library. We divided this cDNA expression library into multiple pools and transfected mammalian cells with the individual pools. Following transfection, we tested the expressed proteins for DNA-binding activity by agarose thin-layer electrophoretic gelshift assay. After we identified a single expression pool for the presence of a DNA-binding protein, the corresponding cDNA pool was further divided into smaller aliquots. Then, the cDNA expression and gelshift clone selection was repeated until a single clone was isolated. In contrast to traditional polyacrylamide gels, the agarose thin-layer is significantly faster and resolves larger DNA-protein complexes. This method can be widely used for the cDNA cloning of DNA- and RNA-binding proteins from various mammalian host cells. |
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| ISSN: | 0736-6205 1940-9818 |