Journal: Human Molecular Genetics

Article Title: Disruption of the methyltransferase-like 23 gene METTL23 causes mild autosomal recessive intellectual disability

doi: 10.1093/hmg/ddu115

Figure Lengend Snippet: Western blot for METTL23 protein: Purified METTL23-His8-tag protein (expressed in E. coli ) was loaded in lane 1 (50 ng) and 2–4 (100 ng). METTL23 proteins were immunoprecipitated from lymphoblastoid cells from healthy control (lane 5) and METTL23-mutant patient PK31 II:1 (lane 6) using polyclonal anti-METTL23. Immunoreactive bands were visualized using ( A ) rabbit polyclonal anti-METTL23-His8-tag antiserum, ( B ) rabbit polyclonal anti-His-tag (C-terminal) antibody and ( C ) sequence-specific anti-METTL23 (C17orf95) antibody as primary antibodies. Arrow (METTL23-His8-tag protein, 22.6 kDa); arrowhead (immunoprecipitated METTL23 protein, 21.5 kDa).

Article Snippet: Membranes were blocked with 5% (w/v) non-fat milk in TBST (Tris-buffered saline containing Tween 20) (25°C, 2 h) and incubated with either (i) rabbit polyclonal anti-METTL23 antiserum (1:2000 in 5%, w/v, BSA), (ii) rabbit polyclonal anti-His-tag (C-terminal) antibody (1:1000 in 5%, w/v, BSA, Relia Tech, Germany) and (iii) rabbit sequence-specific anti-METTL23 (C17orf95) antibody (1:300 in 5%, w/v, BSA, Abgent, Germany, raised against a synthetic peptide; position 137–166 amino acids) (4°C, overnight).

Techniques: Western Blot, Purification, Immunoprecipitation, Control, Mutagenesis, Sequencing