I am trying to charge cysteinyl tRNA with cysteine amino acid and then do a labeling with NBD dye using the thiol reactive group of cysteine. I am not getting any luck with the labeling so far, but i am not sure if the issue is with labeling or the charging aminoacylation reaction. I am using yeast cysteinyl tRNA synthetase enzyme at a conc of 1.5uM per reaction and then run FPLC purification on an anion exchange column. Can you send me a standard protocol which works for cysteine aminoacylation reaction? Any kind of help is greatly appreciated.
I work with E.coli system and use following conditions:
20 mM TrisHCl pH 7.5
20 mM KCl
10 mM MgCl2
25 mM DTT
2 mM ATP
0.5 mM Cys
10 μM tRNACys
1 μM CysRS
CysRS can be removed by acid phenol:chloroform extraction and ATP can be removed using G25 quick spin columns. After that I do EtOH precipitation (at the presence of 0.5 M ammonium acetate, pH 5.2). Most aminoacyl bonds are not stable even at neutral pH, so use slightly acidic buffers whenever is possible. Also Cys can be easily oxidized. To prevent this, I usually add 0.5 mM TCEP in my storage buffer
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