Site-Directed Fluorescence Labeling, TAMRA-C6-AF (amber), CloverDirect

The targeting of unnatural amino acids is genetically defined by selectively introducing UAG amber or CGGG four-base codons into translation templates. For template construction, we recommend expression plasmids from Protein Express (pROX-FL92.1: ProX-tag + His-tag) or BioComber (pURE1-4: no tags). The ProX tag is a special N-terminal peptide designed to promote incorporation of certain large unnatural amino acids requiring precise N-terminal positioning (codon 9) and a specific codon context (flanked by AAC and TCT). Codon 9 of the ProX tag can be varied between UAG, CGGG or TTT (as a control).

During cell-free translation, unnatural amino acid-charged aminoacyl-tRNAs recognize either UAG amber or CGGG codons. If a UAG codon is recognized by release factor 1 (one of three termination factors), protein synthesis will be terminated. If the first three nucleotides of CGGG are recognized as a triplet codon by Arg-tRNA, the reading frame shifts to +1 and a downstream stop codon will terminate protein synthesis. Therefore, full-length translation products will contain unnatural amino acids at 100% efficiency.

Product contents

• Unnatural aminoacyl-tRNA (One tube of unnatural aminoacyl-tRNA is sufficient for 300μ L of in vitro translation reaction.)
  


• tRNA buffer
  

Storage and stability

• Always store this product in the dark.
  


• This product is stable at -70° C until the expiration date which is indicated on the outside of the package.
  


• Once unnatural aminoacyl-tRNA is dissolved in tRNA buffer, it can be stored at -70° C for 2 months.
  

Applications

• Fluorescent labeling
  


• Biotin labeling
  


• PEGylation
  


• Chemical crosslinking
  


• Photo-regulation of a variety of biological processes, including enzyme, receptor, and ion channel activity
  


• In addition, upon request custom services for protein expression with unnatural amino acids are available.