In vitro detection of methylated DNA via recombinant protein MBD2b.
Members of the methyl binding domain (MBD) protein family are known for binding to methylated DNA by recognizing methylated cytosines. Their original function is to regulate protein biosynthesis by recruitment of transcriptional repression complexes to silence gene expression. The aim of the presented work was to detect methylated DNA spotted onto nitrocellulose membranes with recombinant proteins MBD2b, MBD2b-GFP and directly labeled protein MBD2b. Proteins were affinity purified and tested for functionality before application. We were able to show that these functional recombinant proteins bind to unilaterally and symmetrically methylated oligonucleotides and genomic DNA in vitro and thus can be used in various detection assays.
Födermayr
Mathilde
M
Ist Department of Internal Medicine, Elisabethinen Hospital Linz, Fadingerstrasse 1, Linz, Austria. mathilde.foedermayr@elisabethinen.or.at
Pröll
Johannes
J
Zach
Otto
O
Wechselberger
Christian
C
Lutz
Dieter
D
D000906:Antibodies / Q000378:metabolism; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism*; D017422:Sequence Analysis, DNA / Q000379:methods*
D000906:Antibodies; D004268:DNA-Binding Proteins; C115368:MBD2 protein; D011994:Recombinant Proteins