John Mundy, Institute of Molecular Biology, Copenhagen, Denmark
http://www.arabidopsis.org/info/Protocols_Mundy2.jsp#vitrotransl
Use Promega's nuclease-treated -Met (L4210) or -Cys (L4240). The lysate is the same, just the amino acid mixes differ. The Promega protocols work fine as follows, although it is often best to scale them down.
35 ml lysate
7 ml H2O (50 ml final depending on mRNA)
1 ml RNAsin
1 ml 1mM amino acid mixture
2 ml mRNA (1 mg or less)
4 ml Met or Cys (1200Ci/mMol, 10mCi/ml)
inc 37 C 60', place on ice
spot 1 ml on Whatman 1MM, air dry on tin foil
boil in 10% TCA 10', sink filters with ice, rinse 2 x quickly in tap water, once in EtOH and once in acetone, air dry. Count with 5ml Econofluor.
SDS-PAGE ANALYSIS
Take desired volume of mix and dilute to 20 ml w/ water. Add 40ml of cold, saturated AmSO4 (66% final), mix, spin and inc on ice at least 30'. Spin 10' at 4 C, aspirate supernatant completely off and resuspend pellet in SDS sample buffer. This ppt. reduces background significantly. Follow SDS-PAGE protocol (see note for Cys translations).
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