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 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 poolfor 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.
Dobi
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A
Uniformed Services University, Department of Anatomy, Physiology, and Genetics, Bethesda, MD 20814, USA. adobi@usuhs.mil
Debnam
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W
Dalgard
C
C
Owusu
A
A
von Agoston
D
D
D000818:Animals; D002460:Cell Line; D002999:Clone Cells; D018076:DNA, Complementary / Q000302:isolation & purification*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D015870:Gene Expression; D015723:Gene Library; D016601:RNA-Binding Proteins / Q000235:genetics; D051381:Rats; D014157:Transcription Factors / Q000235:genetics; D014162:Transfection
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D016601:RNA-Binding Proteins; D014157:Transcription Factors