Figure 2

DNA affinity precipitation of recombinant and endogenous oocyte extracts proteins. (A) In vitro translated, S³⁵ labeled MBD domain of MeCP2 specifically binds the methylated immobilized probe (SssI+). Conversely, the negative control (TBP2) displays only residual affinity for non-methylated DNA (SssI-). (B) Quantification of DNA methylation-specific binding using the Image-quant software. ~35% of the recombinant MBD domain is recovered after washing and elution from methylated DNA oligos. (C) Western blotting of eluates obtained after 100 mM, 200 mM and 400 mM KCl washes. MeCP2 tolerates 100 mM and 200 mM KCl concentration in the washing buffer whereas most of the MBD3 is eluted with 200 mM salt. (D) DNA affinity precipitation of MBD3, MeCP2 and Mi-2 from oocyte extracts. MeCP2 displays strong preference for methylated DNA, whereas Mi-2 and MBD3 bind both methylated and unmethylated DNA. The short form of MBD3, however, displays a preference for methylated DNA. (E) Establishment of positive (ISWI) and negative (TFIIB) controls for DNA affinity precipitation.