Register now!Newsletter subscription

  • CATALOG PDF Download
  • BROCHURES PDF Download

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 & References

      back to CloverDirect Top


[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]

Site-directly fluorescent-labeled proteins are available to the following analyses.

• Protein 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.

Reference & Citation

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

1) Nakata H, Ohtsuki T, Sisido M.
  A protease inhibitor discovery method using fluorescence correlation spectroscopy with position-specific labeled protein substrates.
  Anal Biochem 15, 121-5 (2009).
2) Abe R, Ohashi H, Iijima I, Ihara M, Takagi H, Hohsaka T, Ueda H.
  "Quenchbodies": quench-based antibody probes that show antigen-dependent fluorescence.
  J Am Chem Soc. 43, 17386-94 (2011).
3) Hiroshima M, Saeki Y, Okada-Hatakeyama M, Sako Y.
  Dynamically varying interactions between heregulin and ErbB proteins detected by single-molecule analysis in living cells.
  Proc Natl Acad Sci USA35, 13984-9 (2012).
4) Nagai H, Ebisu S, Abe R, Goto T, Takahashi N, Hosaka T, Kawada T.
  Development of a novel PPARγ ligand screening system using pinpoint fluorescence-probed protein.
  Biosci Biotechnol Biochem2, 337-41 (2011).
5) Abe R, Jeong HJ, Arakawa D, Dong J, Ohashi H, Kaigome R, Saiki F, Yamane K, Takagi H, Ueda H.
  Ultra Q-bodies: quench-based antibody probes that utilize dye-dye interactions with enhanced antigen-dependent fluorescence.
  Sci Rep. 4, 337-41 (2014). PMID: 24721819
6) Jeong HJ, Ueda H.
  Strategy for making a superior Quenchbody to proteins: effect of the fluorophore position.
  Sensors (Basel). 7, 13285-97 (2014).
7) Tada S, Zang Q, Wang W, Kawamoto M, Liu M, Iwashita M, Uzawa T, Kiga D, Yamamura M, Ito Y.
  In vitro selection of a photoresponsive peptide aptamer to glutathione-immobilized microbeads.
  J Biosci Bioeng 2015 Feb;119(2):137-9. PMID: 25041711
8) Wang W, Uzawa T, Tochio N, Hamatsu J, Hirano Y, Tada S, Saneyoshi H, Kigawa T, Hayashi N, Ito Y, Taiji M, Aigaki T, Ito Y.
  A fluorogenic peptide probe developed by in vitro selection using tRNA carrying a fluorogenic amino acid.
  Chem Commun (Camb) 2014 Mar 18;50(22):2962-4.
9) Tada S, Andou T, Suzuki T, Dohmae N, Kobatake E, Ito Y.
  Genetic PEGylation.
  PLoS One 2012;7(11):e49235.
10) Liu M, Tada S, Ito M, Abe H, Ito Y.
  In vitro selection of a photo-responsive peptide aptamer using ribosome display.
  Chem Commun (Camb) 2012 Dec 18;48(97):11871-3. PMID: 23125981
11) Ezure T, Nanatani K, Sato Y, Suzuki S, Aizawa K, Souma S, Ito M, Hohsaka T, von Heijine G, Utsumi T, Abe K, Ando E, Uozumi N.
  A cell-free translocation system using extracts of cultured insect cells to yield functional membrane proteins.
  PLoS One 2014 Dec 8;9(12):e112874.
12) Goto T, Nagai H, Egawa K, Kim YI, Kato S, Taimatsu A, Sakamoto T, Ebisu S, Hohsaka T, Miyagawa H, Murakami S, Takahashi N, Kawada T.
  Farnesyl pyrophosphate regulates adipocyte functions as an endogenous PPARγ agonist.
  Biochem J. 2011 Aug 15;438(1):111-9.
13) Nagai H, Ebisu S, Abe R, Goto T, Takahashi N, Hosaka T, Kawada T.
  Development of a novel PPARγ ligand screening system using pinpoint fluorescence-probed protein.
  Biosci Biotechnol Biochem 2011;75(2):337-41.

Product List

Site-Directed Fluorescence Labeling
Site-Directed Biotin Labeling
Site-Directed Post-Translational Modification
Site-Directed Unnatural Mutagenesis
Product List [PDF]

*Please note that the delivery lead time may be 2 - 3 months if the product is out of stock. Please inquire about the stock us

To be used for research only. DO NOT use for human gene therapy or clinical diagnosis.