Liu et al. reported a non-radioactive alternative to analyze newly synthesized proteins in cell culture and whole organisms that is based on an alkyne analog of puromycin (Fig. 1). The cell-permeable O-Propargyl-puromycin (Fig. 1A) incorporates into the C-terminus of translating polypeptide chains thereby stopping translation. The resulting C-terminal alkyne labeled proteins can be detected via Cu(I)-catalyzed click chemistry that offers the choice to introduce a Biotin group (Azides of Biotin) for subsequent purification tasks or a fluorescent group (Azides of fluorescent dyes) for subsequent microscopic imaging[1,2,3].
In contrast to previously reported non-radioactive methionine analog-approaches[5,6], methionine free-medium is not required for O-Propargyl-purmoycin-based monitoring of nascent protein synthesis.
Figure 1: O-propargyl-puromycin labels newly synthesized proteins in cell culture and whole organisms (modified according to ). A) Chemical structure of O-Propargyl-puromycin (OP-puro). Visualization of incorporated OP-puro is performed via Cu(I)-catalyzed Click Chemistry (CuAAC) with Azides of fluorescent dyes or Biotin Azides. B) Nascent protein expression in crypts of mouse small intestine was visualized by whole-mount staining. O-Propargyl-puromycin labeled proteins were detected with Tamra-azide (red) and nuclear DNA was stained with OliGreen (green) (modified according to ) C) Newly synthesized proteins in NIH3T3 cells are rapidly detected by incubation with 50 µM OP-puro. OP-puro labeled proteins have been visualized by Alexa568-azide, nuclear DNA was stained with Hoechst dye (modified according to ).
| Liu et al. (2012) Imaging protein synthesis in cells and tissues with an alkyne analog of puromycin. Proc. Natl. Acad. Sci. USA 109(2):413.|
 Goodman et al. (2012) Imaging of protein synthesis with puromycin. Proc Natl Acad Sci USA. 109(17):E989.
 Salic et al. (2012) Reply to Goodman et al.: Imaging protein synthesis with puromycin and the subcellular localization of puromycin-polypeptide conjugates. Proc. Natl. Acad. Sci. USA 109(17):E990.
 Dieterich et al. (2010) In situ visualization and dynamics of newly synthesized proteins in rat hippocampal neurons. Nature Neuroscience 13(7): 897.
 Dieck et al. (2012) Metabolic Labeling with Noncanonical Amino Acids and Visualisation by Chemoselective Fluorescent Tagging. Current Protocols in Cell Biology. 7:7.11.1.
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