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In vitro methylation assay

YA Yoshinori Abe
YS Yosuke Suzuki
KK Kenji Kawamura
NT Nobuyuki Tanaka
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HEK293T cells were transfected with HA-PRMT5 expression plasmids and immunoprecipitated with an anti-HA antibody. A recombinant substrate (5 μg) either GST-GLI1 protein or myelin basic protein (Sigma-Aldrich) as a positive control for a PRMT5 substrate, was incubated with immunoprecipitated HA-PRMT5. S-adenosyl-l-methyl-[3H] methionine (55 Ci mmol−1; Perkin Elmer) was added to each reaction in a total volume of 40 μl MRE, and samples were incubated for 1 h at 30 °C. Samples were separated by SDS–PAGE, and the gel was washed and incubated in amplify solution (Perkin Elmer). The gel was then dried and exposed on BIO-MAX MR X-ray film (Carestream) for 3–7 days at −80 °C.

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