Fluorescent imaging of protein myristoylation during cellular differentiation and development
2017; Elsevier BV; Volume: 58; Issue: 10 Linguagem: Inglês
10.1194/jlr.d074070
ISSN1539-7262
AutoresAndrew J. Witten, Karin F.K. Ejendal, Lindsey Gengelbach O'Brien, Meghan A. Traore, Xu Wang, David M. Umulis, Sarah Calve, Tamara L. Kinzer‐Ursem,
Tópico(s)Cellular transport and secretion
ResumoProtein post-translational modifications (PTMs) serve to give proteins new cellular functions and can influence spatial distribution and enzymatic activity, greatly enriching the complexity of the proteome. Lipidation is a PTM that regulates protein stability, function, and subcellular localization. To complement advances in proteomic identification of lipidated proteins, we have developed a method to image the spatial distribution of proteins that have been co- and post-translationally modified via the addition of myristic acid (Myr) to the N terminus. In this work, we use a Myr analog, 12-azidododecanoic acid (12-ADA), to facilitate fluorescent detection of myristoylated proteins in vitro and in vivo. The azide moiety of 12-ADA does not react to natural biological chemistries, but is selectively reactive with alkyne functionalized fluorescent dyes. We find that the spatial distribution of myristoylated proteins varies dramatically between undifferentiated and differentiated muscle cells in vitro. Further, we demonstrate that our methodology can visualize the distribution of myristoylated proteins in zebrafish muscle in vivo. Selective protein labeling with noncanonical fatty acids, such as 12-ADA, can be used to determine the biological function of myristoylation and other lipid-based PTMs and can be extended to study deregulated protein lipidation in disease states. Protein post-translational modifications (PTMs) serve to give proteins new cellular functions and can influence spatial distribution and enzymatic activity, greatly enriching the complexity of the proteome. Lipidation is a PTM that regulates protein stability, function, and subcellular localization. To complement advances in proteomic identification of lipidated proteins, we have developed a method to image the spatial distribution of proteins that have been co- and post-translationally modified via the addition of myristic acid (Myr) to the N terminus. In this work, we use a Myr analog, 12-azidododecanoic acid (12-ADA), to facilitate fluorescent detection of myristoylated proteins in vitro and in vivo. The azide moiety of 12-ADA does not react to natural biological chemistries, but is selectively reactive with alkyne functionalized fluorescent dyes. We find that the spatial distribution of myristoylated proteins varies dramatically between undifferentiated and differentiated muscle cells in vitro. Further, we demonstrate that our methodology can visualize the distribution of myristoylated proteins in zebrafish muscle in vivo. Selective protein labeling with noncanonical fatty acids, such as 12-ADA, can be used to determine the biological function of myristoylation and other lipid-based PTMs and can be extended to study deregulated protein lipidation in disease states. Recent advancements in chemical biology techniques utilize click chemistry-functionalized biomolecules to study protein regulation in situ. Noncanonical amino acids, sugars, and fatty acids that carry click chemistry functionality can be metabolically incorporated into and onto proteins and have been used to study various aspects of protein biology, including synthesis, turnover, cellular localization, glycosylation, and acylation (1.Zhang X. Zhang Y. Applications of azide-based bioorthogonal click chemistry in glycobiology.Molecules. 2013; 18: 7145-7159Crossref PubMed Scopus (63) Google Scholar, 2.Yuet K.P. Tirrell D.A. Chemical tools for temporally and spatially resolved mass spectrometry-based proteomics.Ann. Biomed. 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For example, the methionine analog, azidohomoalanine, has been shown to be readily incorporated into newly synthesized proteins in vitro in mammalian cell lines (7.Beatty K.E. Liu J.C. Xie F. Dieterich D.C. Schuman E.M. Wang Q. Tirrell D.A. Fluorescence visualization of newly synthesized proteins in mammalian cells.Angew. Chem. Int. Ed. Engl. 2006; 45: 7364-7367Crossref PubMed Scopus (223) Google Scholar, 8.Dieterich D.C. Link A.J. Graumann J. Tirrell D.A. Schuman E.M. Selective identification of newly synthesized proteins in mammalian cells using bioorthogonal noncanonical amino acid tagging (BONCAT).Proc. Natl. Acad. Sci. USA. 2006; 103: 9482-9487Crossref PubMed Scopus (549) Google Scholar) and primary cells (9.Dieterich D.C. Hodas J.J. Gouzer G. Shadrin I.Y. Ngo J.T. Triller A. Tirrell D.A. Schuman E.M. In situ visualization and dynamics of newly synthesized proteins in rat hippocampal neurons.Nat. 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In this way, fluorophores or affinity reagents (e.g. biotin) can be specifically attached to noncanonical biomolecules within complex mixtures in vitro and in vivo for proteomic and imaging analysis. Recent examples of using noncanonical sugars or fatty acids to study post-translational modifications (PTMs) include live-cell imaging of cell membrane fucosylated glycans in developing zebrafish embryos (19.Dehnert K.W. Beahm B.J. Huynh T.T. Baskin J.M. Laughlin S.T. Wang W. Wu P. Amacher S.L. Bertozzi C.R. Metabolic labeling of fucosylated glycans in developing zebrafish.ACS Chem. Biol. 2011; 6: 547-552Crossref PubMed Scopus (80) Google Scholar) and imaging of engineered proteins with lipoic acid ligase in mammalian cells (20.Fernández-Suárez M. Baruah H. Martinez-Hernandez L. Xie K.T. Baskin J.M. Bertozzi C.R. Ting A.Y. Redirecting lipoic acid ligase for cell surface protein labeling with small-molecule probes.Nat. Biotechnol. 2007; 25: 1483-1487Crossref PubMed Scopus (280) Google Scholar). We add to this growing body of work by demonstrating that protein myristoylation can be imaged in situ as a function of time and cellular differentiation using an azide-functionalized myristic acid (Myr) analog, 12-azidododecanoic acid (12-ADA) (outlined in Fig. 1). Myristoylation is the PTM of proteins via the covalent attachment of Myr to the N terminus by the enzyme, N-myristoyltransferase (NMT). Myristoylation plays an essential role in a variety of eukaryotic biological processes, such as targeting proteins to membranes, increasing protein stability, promoting protein-protein interactions, and regulating apoptosis (21.Farazi T.A. Waksman G. Gordon J.I. The biology and enzymology of protein N-myristoylation.J. Biol. Chem. 2001; 276: 39501-39504Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar, 22.Martin D.D. Beauchamp E. Berthiaume L.G. Post-translational myristoylation: fat matters in cellular life and death.Biochimie. 2011; 93: 18-31Crossref PubMed Scopus (151) Google Scholar). Fundamental work by Gordon and colleagues showed, in vitro, that the yeast NMT enzyme can successfully label proteins with noncanonical Myr analogs that contain handles, such as azides, alkynes, and ketones, generating critical click chemistry-based tools that can be utilized to study protein myristoylation (21.Farazi T.A. Waksman G. Gordon J.I. The biology and enzymology of protein N-myristoylation.J. Biol. Chem. 2001; 276: 39501-39504Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar, 23.Speers A.E. Cravatt B.F. Profiling enzyme activities in vivo using click chemistry methods.Chem. Biol. 2004; 11: 535-546Abstract Full Text Full Text PDF PubMed Scopus (650) Google Scholar). Azide- and alkyne-functionalized Myr analogs are incorporated onto naturally myristoylated proteins and proteins engineered to display NMT peptide recognition sequences in recombinant systems coexpressing NMT with high selectivity (6.Ho S.H. Tirrell D.A. Chemoenzymatic labeling of proteins for imaging in bacterial cells.J. Am. Chem. Soc. 2016; 138: 15098-15101Crossref PubMed Scopus (18) Google Scholar, 24.Heal W.P. Wickramasinghe S.R. Bowyer P.W. Holder A.A. Smith D.F. Leatherbarrow R.J. Tate E.W. Site-specific N-terminal labelling of proteins in vitro and in vivo using N-myristoyl transferase and bioorthogonal ligation chemistry.Chem. Commun. (Camb). 2008; 4: 480-482Crossref Google Scholar, 27.Broncel M. Serwa R.A. Ciepla P. Krause E. Dallman M.J. Magee A.I. Tate E.W. Multifunctional reagents for quantitative proteome-wide analysis of protein modification in human cells and dynamic profiling of protein lipidation during vertebrate development.Angew. Chem. Int. Ed. Engl. 2015; 54: 5948-5951Crossref PubMed Scopus (63) Google Scholar). These Myr analogs have also been shown to be metabolically incorporated into mammalian cells in vitro and zebrafish embryos in vivo (27.Broncel M. Serwa R.A. Ciepla P. Krause E. Dallman M.J. Magee A.I. Tate E.W. Multifunctional reagents for quantitative proteome-wide analysis of protein modification in human cells and dynamic profiling of protein lipidation during vertebrate development.Angew. Chem. Int. Ed. Engl. 2015; 54: 5948-5951Crossref PubMed Scopus (63) Google Scholar); making them useful probes for global profiling of the myristoylated proteome. Here, we show that the expression and subcellular localization of 12-ADA-labeled proteins varies dramatically in undifferentiated and differentiated C2C12 skeletal muscle cells. The utility of this method is extended by labeling and visualization of myristoylated proteins within the developing zebrafish embryo using 12-ADA. When combined with proteomic profiling, selective protein labeling using 12-ADA can be used to determine the biological function of myristoylation in different cell states or in response to treatment within discrete time windows; for example, the dysregulation of protein myristoylation in cancer and other diseases (28.Selvakumar P. Lakshmikuttyamma A. Shrivastav A. Das S.B. Dimmock J.R. Sharma R.K. Potential role of N-myristoyltransferase in cancer.Prog. Lipid Res. 2007; 46: 1-36Crossref PubMed Scopus (78) Google Scholar, 29.Wright M.H. Heal W.P. Mann D.J. Tate E.W. Protein myristoylation in health and disease.J. Chem. Biol. 2010; 3: 19-35Crossref PubMed Scopus (181) Google Scholar). Additionally, we expect this method to be readily translatable to visualize lipid-based PTMs in other cellular and in vivo systems. The 12-ADA was synthesized as previously described (26.Kulkarni C. Kinzer-Ursem T.L. Tirrell D.A. Selective functionalization of the protein N terminus with N-myristoyl transferase for bioconjugation in cell lysate.ChemBioChem. 2013; 14: 1958-1962Crossref PubMed Scopus (31) Google Scholar, 30.Devadas B. Lu T. Katoh A. Kishore N.S. Wade A.C. Mehta P.P. Rudnick D.A. Bryant M.L. Adams S.P. Li Q. et al.Substrate specificity of Saccharomyces cerevisiae myristoyl-CoA: protein N-myristoyltransferase. Analysis of fatty acid analogs containing carbonyl groups, nitrogen heteroatoms, and nitrogen heterocycles in an in vitro enzyme assay and subsequent identification of inhibitors of human immunodeficiency virus I replication.J. Biol. Chem. 1992; 267: 7224-7239Abstract Full Text PDF PubMed Google Scholar) with minor modifications (see structure in Fig. 1A). Briefly, a mixture of 12-bromododecanoic acid (1.8 g, 0.0064 mol), sodium azide (1.2 g, 0.019 mol), and 18-crown-6 (0.5 g, 0.0019 mol) were stirred in 25 ml N,N-dimethylformamide under an argon blanket at room temperature overnight. N,N-dimethylformamide was then removed under vacuum. The residue was diluted with 25 ml dichloromethane, followed by the addition of hydrochloric acid (1 M, 25 ml) to quench unreacted sodium azide. The organic layer was rinsed three times with 25 ml water, dried with Na2SO4, filtered, and concentrated under vacuum yielding the desired product (1.5 g, 97% yield) at 95% purity as a pale-yellow liquid. Characterization data for the final product (1H NMR) yielded data that matched published results (30.Devadas B. Lu T. Katoh A. Kishore N.S. Wade A.C. Mehta P.P. Rudnick D.A. Bryant M.L. Adams S.P. Li Q. et al.Substrate specificity of Saccharomyces cerevisiae myristoyl-CoA: protein N-myristoyltransferase. Analysis of fatty acid analogs containing carbonyl groups, nitrogen heteroatoms, and nitrogen heterocycles in an in vitro enzyme assay and subsequent identification of inhibitors of human immunodeficiency virus I replication.J. Biol. Chem. 1992; 267: 7224-7239Abstract Full Text PDF PubMed Google Scholar) 1H NMR 3.19 (t. 2H, J = 6.9 Hz, CH2); 2.28 (t. 2H, J = 7.5 Hz. CH2): 1.53 (m. 4H, 2xCH2); 1.21 (m. 14H) plus 18-crown-6 as a residual impurity 1H NMR 3.51(s. 2H, CH2). C2C12 mouse myoblast cells were obtained from ATCC (catalog number CRL-1772). C2C12 cells were expanded in in growth medium (GM) [DMEM (Thermo Fisher Scientific, Waltham, MA) containing 10% FBS (Corning, Corning, NY), 1% penicillin/streptomycin (Corning), and 1% Glutagro (Corning)] at 37°C and 5% CO2. The medium was changed every 48–72 h and cells were passaged at 80% confluence. For all 12-ADA incorporation experiments, cells between passages five and ten were plated at 1.0 × 104 cells/cm2. To induce myogenic differentiation, confluent cell cultures were incubated with differentiation medium (DM) (DMEM containing 1% penicillin/streptomycin, 1% Glutagro, and 1% FBS). Differentiation was confirmed by visual inspection (after 1–3 days). Confluent myotubes or myoblasts were then incubated with medium containing 100 μM 12-ADA, Myr (Sigma-Aldrich, St. Louis, MO), or vehicle alone (2.8 mM DMSO; Sigma-Aldrich) for 9 h, unless otherwise specified. After incubation with 12-ADA, Myr, or vehicle alone, medium was aspirated and cells were rinsed with 1× PBS. Undifferentiated myoblast cells were harvested with 0.25% (w/v) trypsin and 1 mM EDTA. Cell suspensions were centrifuged at 200 g at 4°C and washed with 1× PBS to remove excess medium. The resultant cell pellets were either stored at −80°C for further use or directly lysed. For differentiated cell cultures, myotubes were isolated using mild trypsinization (0.025% trypsin diluted with 1× PBS) for 5 min, following (31.Schöneich C. Dremina E. Galeva N. Sharov V. Apoptosis in differentiating C2C12 muscle cells selectively targets Bcl-2-deficient myotubes.Apoptosis. 2014; 19: 42-57Crossref PubMed Scopus (39) Google Scholar). The mild trypsinization detaches myotubes leaving only mononuclear myoblasts. This was followed by the normal trypsinization method described above to remove the remaining cells, which were categorized as mid-differentiated myoblasts. Cells were washed and pelleted as described above. Harvested cells were lysed with Mem-PERTM mammalian protein extraction kit (Thermo Fisher Scientific) as per the manufacturer's instructions. Protein concentrations of the soluble cytosolic and membrane fractions were quantified with Pierce 660 nm protein quantitation assay with the Ionic Detergent Compatibility reagent (Thermo Fisher Scientific). The 12-ADA-labeled proteins within the cell protein lysates were selectively labeled with tetramethylrhodamine (TAMRA) alkyne (Click Chemistry Tools, Scottsdale, AZ) (Fig. 1D) using CuAAC (15.Tornøe C.W. Christensen C. Meldal M. Peptidotriazoles on solid phase: [1,2,3]-triazoles by regiospecific copper(i)-catalyzed 1,3-dipolar cycloadditions of terminal alkynes to azides.J. Org. Chem. 2002; 67: 3057-3064Crossref PubMed Scopus (7181) Google Scholar). A Click iT protein reaction buffer kit (Thermo Fisher Scientific) was used per the manufacturer's instructions. A maximum of 60 μg of total protein lysate was added to each click reaction. Free TAMRA-alkyne dye was removed by methanol-chloroform precipitation. Reacted proteins were solubilized in 1× Laemmli sample buffer with 5% β-mercaptoethanol and then boiled at 95°C for 5 min. Protein concentration was quantified with the Pierce 660 nm assay to ensure equal gel loading, and was then resolved by SDS-PAGE on 4–20% polyacrylamide gels (Bio-Rad, Hercules, CA). Gels were scanned for fluorescence on an Azure Biosystems c400 gel imager on the Cy3 channel (526 nm excitation/565 emission) to detect proteins labeled with the TAMRA-alkyne. To quantify the rate of 12-ADA incorporation onto myristoylated proteins, in-gel fluorescence was quantitated using the line tool in ImageJ (National Institutes of Health) and the mean fluorescence intensities for each lane were normalized to the minimum (background) and maximum intensities in each image and plotted as a function of time. Loading consistency was confirmed on the same gel with Coomassie Blue-based GelCode Blue protein stain (Pierce; Thermo Fisher Scientific) and scanned with an Azure Biosystems c400 gel imager. Data were plotted and analyzed using Prism software (GraphPad, La Jolla, CA). Data were fit to a pseudo-first order association curve: y = (max − min) × (1 − e−kt). Where for normalized values max = 1 and min = 0, t is the time of incubation with 12-ADA, and k is the pseudo-first order rate constant (k = 0.22 h−1). C2C12 cells were incubated with 12-ADA, Myr, or DMSO for 6 h, as described above, and then incubated for another 3 h with 12-ADA, Myr, or DMSO media containing 40 μM EdU (Thermo Fisher Scientific). Cells were fixed, permeabilized, and blocked as described above. After blocking, cells were incubated with the following click reaction cocktail for 30 min at room temperature: 2 M Tris (pH 8.5), 50 mM CuSO4, AlexaFluor 488 azide (0.5 mg/ml) (Thermo Fisher Scientific) diluted 1:500, and 0.5 M ascorbic acid, following (32.Salic A. Mitchison T.J. A chemical method for fast and sensitive detection of DNA synthesis in vivo.Proc. Natl. Acad. Sci. USA. 2008; 105: 2415-2420Crossref PubMed Scopus (1316) Google Scholar). Each well was rinsed three times with 1× PBS, incubated in blocking buffer, and then stained with DAPI (to label nuclei, 1:1,000; Sigma-Aldrich) for 10 min at room temperature with slight rocking. Cells were imaged at 5× magnification with a Leica DMI6000 microscope; the number of EdU+ nuclei were normalized by the total number of DAPI-stained nuclei in each frame to determine the percentage of proliferating cells, and averaged over three images per treatment. C2C12 cells were grown to 80% confluence and fed with GM supplemented with 0–40 μM anisomycin (Sigma-Aldrich) for 30 min. Then, 100 μM of 12-ADA were added and cells were incubated for 6 h. Cells were then rinsed with 1× PBS and harvested and lysed with Mem-PERTM mammalian protein extraction kit. The cytosolic lysates were analyzed for 12-ADA incorporation using SDS-PAGE as described above. C2C12 cells were incubated with 100 μM of 12-ADA or Myr for 9 h and were harvested and lysed as described above. Cytosolic proteins containing 12-ADA were labeled with TAMRA-alkyne or AlexaFluor 647-alkyne following (14.Calve S. Witten A.J. Ocken A.R. Kinzer-Ursem T.L. Incorporation of non-canonical amino acids into the developing murine proteome.Sci. Rep. 2016; 6: 32377Crossref PubMed Scopus (55) Google Scholar). Briefly, 60–100 μl of cytosolic protein lysate (50–80 μg total protein, normalized within each experiment) were combined with 10 μl of 400 mM sodium ascorbate for 5 min and then 20 μl of 0.5 M iodoacetamide (VWR, Radnor, PA) were added and incubated for an additional 5 min. Dye-alkyne (0.5 μl 0.8 mM), CuSO4 (16 μl 25 mM) (Sigma-Aldrich), and tris(3-hydroxypropyltriazolylmethyl)amine (40 μl 50 mM) (Click Chemistry Tools) were combined and then added to the protein lysate mixture. After vortexing, 40 μl 100 mM aminoguandine (pH 7) (Sigma-Aldrich) were added and the final mixture was rotated end-over-end for 15 min at room temperature and protected from light. Unreacted dye was removed using methanol-chloroform precipitation and the protein pellets were air dried for at least 30 min. S-acylation was disrupted using hydroxylamine following (33.Martin B.R. Nonradioactive analysis of dynamic protein palmitoylation.Curr. Protoc. Protein Sci. 2013; 73: 14.15.1-14.15.9Crossref Scopus (21) Google Scholar). Pellets from the methanol-chloroform precipitation were resolubilized in 1× Laemmli sample buffer containing 0, 0.3, 0.7, or 1 M aqueous hydroxylamine (Alfa Aesar, Ward Hill, MA) with 5% β-mercaptoethanol. Samples were boiled at 95°C for 5 min and analyzed using SDS-PAGE. In-gel fluorescence was quantitated as described in section "12-ADA incorporation validation via SDS-PAGE." To investigate the effects of hydroxylamine on fluorophore stability, 800 nM AlexaFluor 647-alkyne or TAMRA-alkyne were mixed with 0.01–2 M aqueous hydroxylamine. Samples were analyzed in a 96-well plate for fluorescence on an Azure Biosystems c400 gel imager. Fluorescence was quantified using the line tool in ImageJ (National Institutes of Health) to obtain the mean fluorescent intensities for each well. To assess whether 12-ADA was specifically incorporated at sites of myristoylation, C2C12 cells were incubated with 12-ADA as described above, with the following modifications. For competition with Myr, cells were incubated with 10 μM 12-ADA in combination with increasing concentrations of Myr (1, 10, or 100 μM) for 9 h. To specifically inhibit NMT, cells were preincubated for 30 min with the NMT inhibitor, DDD85646 (Cayman Chemical, Ann Arbor, MI), and then labeled with 10 μM 12-ADA or Myr in the presence of DDD85646 for 6 h. Cells were harvested and the cytosolic fractions were labeled with alkyne fluorophore, as described for the hydroxylamine experiments, except that 12-ADA incorporation was visualized using AlexaFluor 647-alkyne. In-gel fluorescence was quantitated as described in section "12-ADA incorporation validation via SDS-PAGE," with the following modification. Gels were scanned for fluorescence on an Azure Biosystems c400 gel imager on the Cy5 channel (628 nm excitation/676 emission). To visualize 12-ADA incorporation, IBIDI μ-slide Angiogenesis 15-well plates (IBIDI, Fitchburg, WI) were coated with 0.1 mg/ml Matrigel (Corning) diluted in PBS and incubated for 30 min at 37°C incubator. Excess Matrigel medium was removed via aspiration. Cells were seeded between passages five and ten at 5–20 × 104 cells/cm2 and cultured in GM until confluent. Differentiation was induced using DM and was confirmed by visual inspection. Cells were incubated with 12-ADA, Myr, and DMSO alone as described above. Cells were washed with 1× PBS before fixing with 4% paraformaldehyde (diluted in 1× PBS) for 10 min at room temperature. Fixed cells were rinsed with 1× PBS and permeabilized with 0.2% Triton X-100 in 10% donkey serum (diluted in PBS; Lampire Biological Laboratories, Everett, PA) for 15 min at room temperature. Cells were then washed with 1× PBS and blocked with blocking buffer (10% donkey serum in PBS) for 30 min at room temperature and then rinsed with 1× PBS. The 12-ADA-labeled proteins were tagged selectively with TAMRA-alkyne using CuAAC, as described in section "12-ADA incorporation validation via SDS-PAGE" or with TAMRA-dibenzocyclooctyne (DBCO) (Click Chemistry Tools; Fig. 1D) using SPAAC (34.Agard N.J. Prescher J.A. Bertozzi C.R. A strain-promoted [3 + 2] azide-alkyne cycloaddition for covalent modification of biomolecules in living systems.J. Am. Chem. Soc. 2004; 126: 15046-15047Crossref PubMed Scopus (1844) Google Scholar, 35.Agard N.J. Baskin J.M. Prescher J.A. Lo A. Bertozzi C.R. A comparative study of bioorthogonal reactions with azides.ACS Chem. Biol. 2006; 1: 644-648Crossref PubMed Scopus (570) Google Scholar, 36.Yao J.Z. Uttamapinant C. Poloukhtine A. Baskin J.M. Codelli J.A. Sletten E.M. Bertozzi C.R. Popik V.V. Ting A.Y. Fluorophore targeting to cellular proteins via enzyme-mediated azide ligation and strain-promoted cycloaddition.J. Am. Chem. Soc. 2012; 134: 3720-3728Crossref PubMed Scopus (108) Google Scholar), described as follows. Fixed and permeabilized cells were incubated with 20 mM of iodoacetamide for 30 min to react with free thiols, reducing overall background staining (37.van Geel R. Pruijn G.J. van Delft F.L. Boelens W.C. Preventing thiol-yne addition improves the specificity of strain-promoted azide-alkyne cycloaddition.Bioconjug. Chem. 2012; 23: 392-398Crossref PubMed Scopus (204) Google Scholar). The iodoacetamide solution was removed and 10 μM of TAMRA-DBCO were added to each well for 10 min at room temperature and protected from light. Each well was rinsed three times with 1× PBS and then cells were blocked with 10% donkey serum for 5 min before immunocytochemistry. To identify differentiated cells, cultures were incubated with an antibody against skeletal muscle myosin, MY32 (1:100 dilution, ab7784; Abcam, Cambridge, MA) overnight at 4°C. After rinsing with 1× PBS, cells were incubated with AF633 goat anti-mouse IgG2b (1:500, A-21050; Thermo Fisher Scientific) and DAPI (to label nuclei, 1:1,000; Sigma-Aldrich) for 1 h at room temperature. Antibodies were diluted in blocking buffer and all staining steps were protected from light. Labeled cells were kept hydrated in 1× PBS and imaged on a Leica DMI6000 microscope. Images were processed and analyzed using Fiji 2.0v software (National Institutes of Health). Zebrafish, including adult fish and embryos, were maintained according to the Purdue Animal Care and Use Committee protocol (#1501001180) at Purdue University. To ensure that 12-ADA would be taken up by the embryos, 18 h post fertilization (hpf) zebrafish were dechorionated using pronase (Sigma) diluted in E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl, and 0.33 mM MgSO4 in milliQ water) and incubated overnight at 27°C. At 24 hpf, embryos were rinsed with 1× PBS and transferred to a clean specimen dish filled with 20 μM 12-ADA diluted in E3. The larvae were incubated at 27°C for 12 h. After incubation, larvae were rinsed in 0.1% PBST (1× PBS + 0.1% Tween-20) and fixed with 4% paraformaldehyde in 1× PBS for 2 h at room temperature. Embryos were either stained immediately or dehydrated in methanol and stored at −20°C. After fixation or rehydration, the zebrafish larvae were washed twice in a PBDT solution (PBST + 1% DMSO). They were then washed briefly in PBS and permeabilized for 20 min in 10 μg/ml proteinase K (Roche) in PBST. Larvae were washed four times for 5 min with PBST. To prevent nonspecific binding of staining reagents, larvae were incubated in a blocking solution made of 5% BSA (Sigma-Aldrich) and 10% goat serum (Lampire) in PBST for a minimum of 3 h at 4°C or 2 h at room temperature. Larvae were then rinsed briefly in PBS. To identify 12-ADA-labeled proteins, larvae were incubated (when indicated) in the following CuAAC reaction cocktail [as described in (13.Hinz F.I. Dieterich D.C. Tirrell D.A. Schuman E.M. Non-canonical amino acid labe
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