TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol provides a detailed description of how to design and perform BONCAT experiments using two different bioorthogonal amino acids, <span class="s1">L</span>-azidohomoalanine (AHA) and <span class="s1">L</span>-homopropargylglycine (HPG), which are both surrogates of <span class="s1">L</span>-methionine. It illustrates how incorporation of these noncanonical amino acids into new proteins can be detected in whole cells via copper-catalyzed or strain-promoted azide-alkyne click chemistry and outlines how the visualization of translational activity can be combined with the taxonomic identification of cells via FISH. Last, the protocol discusses potential problems that might be encountered during BONCAT experiments with environmental samples and how they can be overcome.</p>
<p> </p>
<p>Book chapter describing this BONCAT protocol is published in: </p>
<p><strong>Detection of Protein-Synthesizing Microorganisms in the Environment via Bioorthogonal Noncanonical Amino Acid Tagging (BONCAT) </strong></p>
<p><strong> </strong></p>
<p><strong>Authors: Roland Hatzenpichler and Victoria J. Orphan</strong></p>
<p>T.J. McGenity et al. (eds.), Hydrocarbon and Lipid Microbiology Protocols, Springer Protocols Handbooks, DOI 10.1007/8623_2015_61, © Springer-Verlag Berlin Heidelberg 2015</p>

[BEFORE_START]
<p>There are two variants of azide-alkyne click reactions:</p>
<p>(1) a Cu (I)-catalyzed cycloaddition reaction and</p>
<p>(2) a strain-promoted version that exploits the high reactivity of a cyclooctyne system that allows the click reaction to take place in the absence of a catalyst.</p>
<p> </p>
<p>Both labeling reactions are simple with only a few inexpensive chemicals required. The protocol can be preformed within 1–2 hours to identify transcriptionally active microorganisms in a variety of environmental samples after incubation with a methionine surrogate.  In our experience, the BONCAT reaction is generally not influenced by the presence of complex organic (e.g., an extracellular matrix) or inorganic (e.g., minerals or sediment particles) substances. This results in a low level of background noise when applied to environmental samples. While copper-catalyzed click chemistry can be employed to detect both AHA- and HPG-tagged proteins, the strain-promoted click reaction is restricted to the visualization of AHA uptake (because HPG does not contain an azide group).<br /><br />Solutions required for BONCAT labeling (Unless indicated otherwise, all reagents can be stored at room temperature).</p>
<p><br />For L-2-amino-4-azidobutanoic acid (L-azidohomoalanine, AHA): Dissolve in nano-pure water, adjust to pH 7.0, filter sterilize (0.2 μm), and store in the dark at 4ºC. Prepare stock solutions (typically in the range between 1–100 mM).<br /><br />For L-2-amino-5-hexynoic acid (L-homopropargylglycine, HPG): Dissolve in nano-pure water, adjust to pH 7.0, filter sterilize (0.2 μm), and store in the dark at 4ºC. Prepare stock solutions (1–100 mM).<br /><br /></p>
<p>Once samples have been incubated with either HPG or AHA, paraformaldehyde fixation (PFA) and preparation of microbial cells on a teflon coated slide or polycarbonate filter should be done following a standard protocol for FISH (e.g. Ishii et al. 2004 or Daims et al 2005).  After PFA fixed samples are prepared, the copper-catalyzed (Cu I) click reaction for fluorescence detection of newly synthesized proteins in whole cells requires the following solutions:</p>
<p> </p>
<p>Phosphate-buffered saline (PBS): 130 mM NaCl, 5% (v/v) phosphate buffer in nano-pure water, adjust to pH 7.4, and sterilize filter (0.2 μm).</p>
<p> </p>
<p>20 mM Copper sulfate solution (CuSO4  5 H2O) in nano-pure water, sterilize filter (0.2 μm), and store at room temperature or 4ºC.</p>
<p> </p>
<p>50 mM THPTA : Tris[(1-hydroxypropyl-1H-1,2,3-triazol-4-yl)methyl]amine in nano-pure water, sterilize filter (0.2 μm), and store in aliquots at -20ºC.</p>
<p> </p>
<p>100 mM freshly prepared sodium ascorbate in 1X PBS (this reducing agent is added to ensure the copper sulfate solution has a sufficient amount of copper I to catalyze the azide-alkyne reaction). Should be made fresh each time and used the same day.</p>
<p> </p>
<p>100 mM Aminoguanidine hydrochloride in 1X PBS (this reagent should also be made fresh each time)</p>
<p> </p>
<p>1–10 mM stock solutions in either dimethylsulfoxide (DMSO) or dimethylformamide (DMF) for preparation of the dye solution following manufacturer's instructions (Alexa dyes or Cy dyes)</p>
<p>for HPG incubations, use azide-conjugated fluorophores</p>
<p>for AHA incubations, use alkyne-conjugated fluorophores</p>
<p> </p>
<p>See following references for additional information on copper-catalyzed and strain promoted Click and FISH sample preparation.</p>
<p> </p>
<p>Ishii, K., M. Mußmann, B. J. MacGregor, and R. Amann. 2004. An improved fluorescence in situ hybridization protocol for the identification of bacteria and archaea in marine sediments. FEMS Microbiology Ecology 50:203-212.</p>
<p> </p>
<p>Daims H, Stoecker K, Wagner M (2005) Fluorescence in situ hybridization for the detection of prokaryotes. In: Osborn AM, Smith CJ (eds) Molecular microbial ecology. Bios Advanced Methods, Abingdon, pp 213–239</p>
<p> </p>
<p>Bagert JD, Xie YJ, Sweredoski MJ, Qi Y, Hess S, Schuman EM, Tirrell DA (2014) Quantitative, time-resolved proteomic analysis by combining bioorthogonal noncanonical amino acid tagging and pulsed stable isotope labeling by amino acids in cell culture. Mol Cell Proteomics 13(5):1352–1358.</p>
<p> </p>
<p>Dieterich DC, Link AJ, Graumann J, Tirrell DA, Schuman EM (2006) Selective identification of newly synthesized proteins in mammalian cells using bioorthogonal noncanonical amino acid tagging (BONCAT). Proc Natl Acad Sci U S A 103:9482–9487</p>
<p> </p>
<p>Torne CW, Christensen C, Meldal M (2002) Peptidotriazoles on solid phase: [1,2,3]- friazoles by regiospecific copper(I)-catalyzed 1,3-dipolar cycloadditions of terminal alkynes to azides. J Org Chem 67:3057–3064</p>
<p> </p>
<p>Agard NJ, Prescher JA, Bertozzi CR (2004) A strain-promoted [3 + 2] azide-alkyne cycloaddition for covalent modification of biomolecules in living systems. J Am Chem Soc 126:15046–15047</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.