Immunoblotting. Affinity Purified Rabbit anti-DNMT3L was used at a 1:1,500 dilution to detect human DNMT3L by western blot after immuno-precipitation using the same antibody. Transfection of U2OS cells (10cm dish) was accomplished using 3 ug of GAL4-DNMT3L. Protein extraction using IPH at 150mM. For IP and WB conditions see Fuks et al.(2000)..,.For western blotting the antibody was used at 0.4ug/ml in TBS milk 2%, BSA 0.5%. Detection occurred using ECL. Note: this antibody.,.works for western blotting only following immunoprecipitation. We have not had good success using the antibody directly for western blotting. The expected molecular weight of human DNMT3L is 43.6 kDa.

Schematic representation of the Dnmt3 family members. The conserved N-terminal cysteine-rich region, the PHD-like motif, characteristic of the Dnmt3 family is shown. The C-terminal domain is the catalytic methyltransferase domain present in Dnmt3a and Dnmt3b whereas the key catalytic motifs are absent in Dnmt3L. Numbers indicate amino acid residues.