Fluorescent labeling of cell-free synthesized proteins with fluorophore-conjugated methionylated tRNA derived from in vitro transcribed tRNA.
A simple and practical method for preparing fluorophore-conjugated methionylated tRNA necessary for tRNA-mediated fluorescent labeling of cell-free synthesized proteins was developed. Without complicated chromatographic purification and subsequent concentration, fluorophore-conjugated methionylated tRNA with higher purity and fluorescence concentration could be synthesized from in vitro transcribed tRNA instead of from a total tRNA mixture, which has been routinely used as a tRNA source. Although fluorophore-conjugated methionylated tRNA derived from in vitro transcribed tRNA was purified by simple phenol extraction following alcohol precipitation, it worked well in tRNA-mediated fluorescent labeling, yielding an improved signal-to-noise ratio and higher fluorescence intensity compared to the conventional total tRNA-based method. Based on its simplicity in the preparation of labeling agent with higher purity and fluorescence concentration, the developed method will accelerate the prevalence of fluorescence-based detection of cell-free synthesized proteins.
Jun
Soo Youn
SY
Department of Life Science, College of Natural Science, Chung-Ang University, Seoul 156-756, South Korea.
Kang
Sang Hyeon
SH
Lee
Kwang-Ho
KH
D005453:Fluorescence; D008715:Methionine / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D012358:RNA, Transfer, Met / Q000302:isolation & purification / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods*; D014158:Transcription, Genetic
D011506:Proteins; D012358:RNA, Transfer, Met; D008715:Methionine