In vitro translation

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)

inc37℃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' at4℃ 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).