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Pin-point Protein Labeling An amber stop codon (UAG) is used for pin-point fluorescence or biotin labeling.

CloverDirect™ tRNA Reagents for Site-Directed Protein Functionalization_Application


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Application

[Expression of site-directly labeled proteins]

Fluorescent- and biotin-labeled unnatural amino acids are incorporated into eight prokaryote and eukaryote proteins. The site-directly fluorescent-labeled proteins can be visualized on SDS-PAGE using a laser-based fluorescence scanner. The proteins are also detectable by an antibody against tag peptide or biotin. A 0.25 ? 1 µL of translational reaction mixture is sufficient for the detection. · Labeled unnatural amino acids that are not incorporated into proteins are detected at the bottom of SDS-PAGE gel.

2UAG : UAG codon is inserted after initiator AUG codon.
ProX tag : ProX tag is fused to the N-terminus.
Applied volume: 0.25 µL of translational reaction mix
Fluorescence image (Top) are visualized with Ex and Em
wavelengths listed below:
HiLyteFluor488 Ex : 488nm / Em : 520 nm
TAMRA Ex : 532nm / Em : 580 nm
ATTO633 Ex : 635nm / Em : 670 nm
Western blotting (Bottom) are visualized by anti-His tag antibody (for fluorescent amino acids) and anti-biotin antibody (for biotin).

[Applications for site-directly fluorescent labeled proteins]

The site-directly fluorescent-labeled proteins are available to the following measurements.

·Interaction analysis using single molecule fluorescence analysis Single Molecule fluorescence detection system (MF20 / Fluor Point-Light MF20; OLYMPUS)
·Conformation analysis of protein by inter- or intra-molecular fluorescence resonance energy transfer (FRET)
·Functional analysis in cell imaging
·Interaction analysis in protein array
·Expression analysis by in-gel fluorescent detection of SDS-PAGE

<References>
1. FRET analysis of protein conformational change through position-specific incorporation of fluorescent amino acids Daisuke Kajihara, Ryoji Abe, Issei Iijima, Chie Komiyama, Masahiko Sisido, Takahiro Hohsaka
Nature Methods., 3, 923-929 (2006).
2. Position-specific incorporation of biotinylated non-natural amino acids into a protein in a cell-free translation system Takayoshi Watanabe, Norihito Muranaka, Issei Iijima, Takahiro Hohsaka
Biochem. Biophys. Res. Commun., 361, 794-799 (2007)
3. Comprehensive screening of amber suppressor tRNAs suitable for incorporation of non-natural amino acids in a cell-free translation system
Hikaru Taira, Yosuke Matsushita, Kenji Kojima, Kaori Shiraga, Takahiro Hohsaka
Biochem. Biophys. Res. Commun., 374, 304-308 (2008).
4. Efficient Incorporation of Nonnatural Amino Acids with Large Aromatic Groups into Streptavidin in in vitro Protein Synthesizing Systems
Takahiro Hohsaka, Daisuke Kajihara, Yuki Ashizuka, Hiroshi Murakami, Masahiko Sisido
J. Am. Chem. Soc., 121, 34-40 (1999).

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