Name: efficient and site-specific antibody labeling by strain-promoted azide-alkyne cycloaddition
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Description: Watch this Scientific Journal Video about Efficient and Site-specific Antibody Labeling by Strain-promoted Azide-alkyne Cycloaddition at JoVE.com

1. Costruzione plasmide <ol><li> Costruire un plasmide di espressione (pBAD-HerFab-L177TAG) che esprimesse il gene bersaglio degli anticorpi (pBAD-HerFab-WT) con il suo 6 -tag, e sostituire il codone per la leucina-177 con il codone ambra (TAG) 27, utilizzando convenzionale tecnica di mutagenesi sito-diretta. Vedere la Tabella dei Materiali. </li><li> Costruire un altro plasmide di espressione (pEVOL-AFRS) contenente i geni per il tRNA evoluta Tyr e-tRNA sinteta...

<ol>
	<li>Wang, L., Schultz, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanding+the+genetic+code.">Expanding the genetic code.</a> Angew. Chem. Int. Ed. 44 (1), 34-66 (2004).</li><li>Wu, X., Schultz, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+at+the+interface+of+chemistry+and+biology.">Synthesis at the interface of chemistry and biology.</a> J. Am. Chem. Soc. 131 (35), 12497-12515 (2009).</li><li>Liu, C. C., Schultz, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adding+new+chemistries+to+the+genetic+code.">Adding new chemistries to the genetic code.</a> Annu. Rev. Biochem. 79, 413-444 (2010).</li><li>Wang, L., Zhang, Z., Brock, A., Schultz, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Addition+of+the+keto+functional+groupto+the+genetic+code+of+Escherichia+coli.">Addition of the keto functional groupto the genetic code of Escherichia coli.</a> proc. Natl. Acad. Sci. U.S.A. 100 (1), 56-61 (2003).</li><li>Chin, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Addition+of+p-azido-L-phenylalanine+to+the+genetic+code+of+Escherichia+coli.">Addition of p-azido-L-phenylalanine to the genetic code of Escherichia coli.</a> J. Am. Chem. Soc. 124 (31), 9026-9027 (2002).</li><li>Deiters, A., Schultz, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+incorporation+of+an+alkyne+into+proteins+in+Esshcerichia+coli.">In vivo incorporation of an alkyne into proteins in Esshcerichia coli.</a> Bioorg. Med. Chem. Lett. 15 (5), 1521-1524 (2005).</li><li>Plass, T., Milles, S., Koehler, C., Schultz, C., Lemke, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+encoded+copper-free+click+chemistry.">Genetically encoded copper-free click chemistry.</a> Angew. Chem. Int. Ed. 50 (17), 3878-3881 (2011).</li><li>Seitchik, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+encoded+tetrazine+amino+acid+directs+rapid+site-specific+in+vivo+bioorthogonal+ligation+with+transcyclooctenes.">Genetically encoded tetrazine amino acid directs rapid site-specific in vivo bioorthogonal ligation with transcyclooctenes.</a> J. Am. Chem. Soc. 134 (6), 2898-2901 (2014).</li><li>Lee, Y. -. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetically+encoded+acrylamide+fuctionality.">A genetically encoded acrylamide fuctionality.</a> ACS Chem. Biol. 8 (8), 1664-1670 (2013).</li><li>Lang, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+encoded+norbornene+directs+site-specific+cellular+protein+labelling+via+a+rapid+bioorthogonal+raction.">Genetically encoded norbornene directs site-specific cellular protein labelling via a rapid bioorthogonal raction.</a> Nat Chem. 4 (4), 298-304 (2012).</li><li>Lang, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+encoding+of+bicyclononynes+and+trans-cyclooctenes+for+site-specific+protein+labeling+in+vitro+and+in+live+mammalian+cells+via+rapid+fluorogenic+Diels-Alder+reactions.">Genetic encoding of bicyclononynes and trans-cyclooctenes for site-specific protein labeling in vitro and in live mammalian cells via rapid fluorogenic Diels-Alder reactions.</a> J. Am. Chem. Soc. 134 (25), 10317-10320 (2012).</li><li>Jewett, J. C., Sletten, E. M., Bertozzi, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Cu-free+click+chemistry+with+readily+synthesized+biarylazacyclooctynones.">Rapid Cu-free click chemistry with readily synthesized biarylazacyclooctynones.</a> J. Am. Chem. Soc. 132 (11), 3688-3690 (2010).</li><li>Debets, M. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Azadibenzocyclooctynes+for+fast+and+efficient+enzyme+PEGylation+via+copper-free+(3+++2)+cycloaddition.">Azadibenzocyclooctynes for fast and efficient enzyme PEGylation via copper-free (3 + 2) cycloaddition.</a> Chem. Commun. 46 (1), 97-99 (2010).</li><li>Sletten, E. M., Bertozzi, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+mechanism+to+mouse:+a+tale+of+two+bioorthogonal+reactions.">From mechanism to mouse: a tale of two bioorthogonal reactions.</a> Acc. Chem. Res. 44 (9), 666-676 (2011).</li><li>Debets, M. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioconjugation+with+strained+alkenes+and+alkynes.">Bioconjugation with strained alkenes and alkynes.</a> Acc. Chem. Res. 44 (9), 805-815 (2011).</li><li>Mbua, N. E., Guo, J., Wolfert, M. A., Steet, R., Boons, G. -. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain-promoted+alkyne&#8722;azide+cycloadditions+(SPAAC)+reveal+new+features+of+glycoconjugate+biosynthesis.">Strain-promoted alkyne&#8722;azide cycloadditions (SPAAC) reveal new features of glycoconjugate biosynthesis.</a> ChemBioChem. 12 (12), 1912-1921 (2011).</li><li>Kuzmin, A., Poloukhtine, A., Wolfert, M. A., Popik, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+functionalization+using+catalyst-free+azide-alkyne+cycloaddition.">Surface functionalization using catalyst-free azide-alkyne cycloaddition.</a> Bioconjug. Chem. 21 (11), 2076-2085 (2011).</li><li>Yao, J. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorophore+targeting+to+cellular+proteins+via+enzymemediated+azide+ligation+and+strain-promoted+cycloaddition.">Fluorophore targeting to cellular proteins via enzymemediated azide ligation and strain-promoted cycloaddition.</a> J. Am. Chem. Soc. 134 (8), 3720-3728 (2012).</li><li>Hao, Z., Hong, S., Chen, X., Chen, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introducing+bioorthogonal+functionalities+into+proteins+in+living+cells.">Introducing bioorthogonal functionalities into proteins in living cells.</a> Acc. Chem. Res. 44 (9), 742-751 (2011).</li><li>Reddington, S. C., Tippmann, E. M., Jones, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Residue+choice+defines+efficiency+and+influence+of+bioorthogonal+protein+modification+via+genetically+encoded+strain+promoted+Click+chemistry.">Residue choice defines efficiency and influence of bioorthogonal protein modification via genetically encoded strain promoted Click chemistry.</a> Chem. Commun. 48 (9), 8419-8421 (2012).</li><li>Chari, R. V. J., Miller, M. L., Widdison, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody-drug+conjugates:+an+emerging+concept+in+cancer+therapy.">Antibody-drug conjugates: an emerging concept in cancer therapy.</a> Angew. Chem. Int. Ed. 53 (15), 3751-4005 (2014).</li><li>Tsao, M., Tian, F., Schultz, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+staudinger+modification+of+proteins+containing+p-Azidophenylalanine.">Selective staudinger modification of proteins containing p-Azidophenylalanine.</a> ChemBioChem. 6 (12), 2147-2149 (2005).</li><li>Brustad, E. M., Lemke, E. A., Schultz, P. G., Deniz, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+general+and+efficient+method+for+the+site-specific+dual-labeling+of+proteins+for+single+molecule+fluorescence+resonance+energy+transfer.">A general and efficient method for the site-specific dual-labeling of proteins for single molecule fluorescence resonance energy transfer.</a> J. Am. Chem. Soc. 130 (52), 17664-17665 (2008).</li><li>Milles, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Click+strategies+for+single-molecule+protein+fluorescence.">Click strategies for single-molecule protein fluorescence.</a> J. Am. Chem. Soc. 134 (11), 5187-5195 (2012).</li><li>Jang, S., Sachin, K., Lee, h. J., Kim, D. W., Lee, h. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+simple+method+for+protein+conjugation+by+copper-free+click+reaction+and+its+application+to+antibody-free+Western+blot+analysis.">Development of a simple method for protein conjugation by copper-free click reaction and its application to antibody-free Western blot analysis.</a> Bioconjug. Chem. 23 (11), 2256-2261 (2012).</li><li>Kazane, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-assembled+antibody+multimers+through+peptide+nucleic+acid+conjugation.">Self-assembled antibody multimers through peptide nucleic acid conjugation.</a> J. Am. Chem. Soc. 135 (1), 340-346 (2012).</li><li>Ko, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+and+site-specific+antibody+labeling+by+strain-promoted+azide-alkyne+cycloaddition.">Efficient and site-specific antibody labeling by strain-promoted azide-alkyne cycloaddition.</a> Bull. Korean Chem. Soc. 36 (9), 2352-2354 (2015).</li><li>Young, T. S., Ahmad, I., Yin, J. A., Schultz, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+enhanced+system+for+unnatural+amino+acid+mutagenesis+in+E.+coli.">An enhanced system for unnatural amino acid mutagenesis in E. coli.</a> J. Mol. Biol. 395 (2), 361-374 (2010).</li><li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Anal. Biochem. 72, 248-254 (1976).</li><li>Cho, H. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+extracellular+region+of+HER2+alone+and+in+complex+with+the+Herceptin+Fab.">Structure of the extracellular region of HER2 alone and in complex with the Herceptin Fab.</a> Nature. 421 (6924), 756-760 (2003).</li><li>Kolb, H. C., Finn, M. G., Sharpless, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Click+chemistry+:+Diverse+chemical+function+from+a+few+good+reactions.">Click chemistry : Diverse chemical function from a few good reactions.</a> Angew. Chem. Int. Ed. 40 (11), 2004-2021 (2001).</li></ol>

L&#39;incorporazione genetica di aminoacidi non naturali in proteine ​​ha diversi vantaggi rispetto ad altri metodi utilizzati per la modifica della proteina. 1-3 Uno dei vantaggi importanti è la sua applicabilità generale a qualsiasi tipo di proteina. In linea di principio, non vi è alcuna limitazione nella scelta di una proteina bersaglio e un sito target della proteina. Tuttavia, la sostituzione di un residuo strutturalmente o funzionalmente importante con SAU può comportare alterando la struttura ...

Dal momento che la costituzione genetica di p -methoxyphenylalanine in Escherichia coli è stato segnalato, 1 più di 100 amminoacidi non naturali (UAAs) sono stati inseriti con successo in varie proteine. 1-3 Tra questi UAAs, gli aminoacidi contenenti gruppi funzionali bioorthogonal sono stati ampiamente studiati e rappresentano la percentuale più elevata. I gruppi funzionali bioorthogonal utilizzati nei UAAs includono chetone, 4 azide, 5 alchino, 6 cyclooctyne, 7 tetrazina, 8 α, β-insaturo ammide, 9 norbonene, 10 transcyclooctene, 11 e biciclo [6.1.0] -nonyne. 11 Sebbene ogni gruppo funzionale ha i suoi vantaggi e svantaggi, gli amminoacidi azide contenenti più sono stati ampiamente utilizzati per la coniugazione di proteine. p -Azidophenylalanine (AF), uno degli amminoacidi azido-contenente, è prontamente disponibile, e la sua incorporazione efficiency è eccellente. proteine ​​mutanti contenenti questo aminoacido può essere fatto reagire con alchini di cicloaddizione di rame-catalizzata o con cyclooctynes ​​di SPAAC. 12-20 Recentemente, biofarmaceutici hanno attratto grande attenzione nel settore farmaceutico. Il coniugato anticorpo-farmaco (ADC) è una classe di anticorpi terapeutici che sono vantaggiosi per la loro capacità di terapia mirata per il trattamento dei tumori umani 21 e altre malattie. Più di 50 ADC sono attualmente in sperimentazione clinica, e il numero è in rapido aumento. Nello sviluppo di ADC, molti fattori devono essere considerati per massimizzare l&#39;efficacia e minimizzare gli effetti collaterali. Tra questi fattori, una reazione di coniugazione efficiente e sito-specifica per formare un legame covalente tra un anticorpo ed un farmaco è critica. L&#39;efficienza desiderata e specificità nella reazione di coniugazione può essere ottenuto mediante coniugazione con un gruppo funzionale in un bioorthogonalamminoacido non naturale che è specificamente incorporata in un anticorpo. 22-26 Qui, si segnala un protocollo al sito-specifico incorporano AF in un frammento di anticorpo e coniugare il frammento di anticorpo mutante con una sonda biochimico.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1. plasmid Construction</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>plasmid pBAD_HerFab_L177TAG</td>
			<td></td>
			<td></td>
			<td>optionally contain the amber stop codon(TAG) at a desired position. Ko, W. et al. Efficient and Site-Specific Antibody Labeling by Strain-promoted Azide-Alkyne Cycloaddition. BKCS. 36 (9), 2352-2354, doi: 10.1002/bkcs.10423, (2015)</td>
		</tr>
		<tr>
			<td>plasmid pEvol-AFRS</td>
			<td></td>
			<td></td>
			<td>Young, T. S., Ahmad, I., Yin, J. A., and Schultz, P. G. An enhanced system for unnatural amino acid mutagenesis in E. coli. J. Mol. Biol. 395 (2), 361-374, doi: 10.1016/j.jmb.2009.10.030, (2010)</td>
		</tr>
		<tr>
			<td>DH10B</td>
			<td>Invitrogen</td>
			<td>C6400-03</td>
			<td>Expression Host</td>
		</tr>
		<tr>
			<td>Plasmid Mini-prep kit</td>
			<td>Nucleogen</td>
			<td>5112</td>
			<td>200/pack</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Intron biotechnology</td>
			<td>32034</td>
			<td>500g</td>
		</tr>
		<tr>
			<td>Ethidium bromide</td>
			<td>Alfa Aesar</td>
			<td>L07482</td>
			<td>1g</td>
		</tr>
		<tr>
			<td>LB Broth</td>
			<td>BD Difco</td>
			<td>244620</td>
			<td>500g</td>
		</tr>
		<tr>
			<td>2. Culture preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2.1) Electroporation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro pulser&#160;</td>
			<td>BIO-RAD</td>
			<td>165-2100</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro pulser cuvette</td>
			<td>BIO-RAD</td>
			<td>165-2089</td>
			<td>0.1cm electrode gap, pkg. of 50</td>
		</tr>
		<tr>
			<td>Ampicillin Sodium</td>
			<td>Wako</td>
			<td>018-10372</td>
			<td>25g</td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>Alfa Aesar</td>
			<td>B20841</td>
			<td>25g</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>SAMCHUN</td>
			<td>214230</td>
			<td>500g</td>
		</tr>
		<tr>
			<td>SOC medium</td>
			<td>Sigma</td>
			<td>S1797</td>
			<td>100ML</td>
		</tr>
		<tr>
			<td>3. Expression and purification of HerFab-L177AF</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3.1 Expression of Herfab-L177AF</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>p-azido-L-phenylalanine (AF)</td>
			<td>Bachem</td>
			<td>F-3075.0001</td>
			<td>1g</td>
		</tr>
		<tr>
			<td>L(+)-Arabinose, 99%</td>
			<td>Acros</td>
			<td>104981000</td>
			<td>100g</td>
		</tr>
		<tr>
			<td>Hydrochloric acid, 35~37%</td>
			<td>SAMCHUN</td>
			<td>H0256</td>
			<td>500ml</td>
		</tr>
		<tr>
			<td>3.2 Cell lysis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tris(hydroxymethyl)aminomethane, 99%</td>
			<td>SAMCHUN</td>
			<td>T1351</td>
			<td>500g</td>
		</tr>
		<tr>
			<td>EDTA disodium salt dihydrate, 99.5%</td>
			<td>SAMCHUN</td>
			<td>E0064</td>
			<td>1kg</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S9378</td>
			<td>500g</td>
		</tr>
		<tr>
			<td>Lysozyme</td>
			<td>Siyaku</td>
			<td>126-0671</td>
			<td>1g</td>
		</tr>
		<tr>
			<td>3.3 Ni-NTA Affinity Chromatography</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ni-NTA resin</td>
			<td>QIAGEN</td>
			<td>30210</td>
			<td>25ml</td>
		</tr>
		<tr>
			<td>Polypropylene column</td>
			<td>QIAGEN</td>
			<td>34924</td>
			<td>50/pack, 1ml capacity</td>
		</tr>
		<tr>
			<td>Imidazole, 99%</td>
			<td>SAMCHUN</td>
			<td>I0578</td>
			<td>1kg</td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic, 98%</td>
			<td>SAMCHUN</td>
			<td>S0919</td>
			<td>1kg</td>
		</tr>
		<tr>
			<td>Sodium Chloride, 99%</td>
			<td>SAMCHUN</td>
			<td>S2907</td>
			<td>1kg</td>
		</tr>
		<tr>
			<td>4. Conjugation of Purified HerFab-L177AF with Alkyne Probes Using Strain-Promoted Azide-Alkyne Cycloaddition (SPAAC)&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cy5.5-ADIBO&#160;</td>
			<td>FutureChem</td>
			<td>FC-6119</td>
			<td>1mg</td>
		</tr>
		<tr>
			<td>5. Purification of Labeled HerFab</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra 0.5 mL Centrifugal Filters</td>
			<td>MILLIPORE</td>
			<td>UFC500396</td>
			<td>96/pack, 500ul capacity</td>
		</tr>
		<tr>
			<td>6. SDS-PAGE Analysis of Labeled HerFab and Fluorescent Gel Scanning</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1,4-Dithio-DL-threitol, DTT, 99.5 %</td>
			<td>Sigma</td>
			<td>10708984001</td>
			<td>10g</td>
		</tr>
		<tr>
			<td>NuPAGE LDS Sample Buffer, 4X</td>
			<td>Thermofisher</td>
			<td>NP0007</td>
			<td>10ml</td>
		</tr>
		<tr>
			<td>MES running buffer</td>
			<td>Thermofisher</td>
			<td>NP0002</td>
			<td>500ml</td>
		</tr>
		<tr>
			<td>Nupage Novex 4-12% SDS PAGE gels</td>
			<td>Thermofisher</td>
			<td>NO0321</td>
			<td>12well</td>
		</tr>
		<tr>
			<td>Coomassie Brilliant Blue R-250</td>
			<td>Wako</td>
			<td>031-17922</td>
			<td>25g</td>
		</tr>
		<tr>
			<td>Typhoon 9210 variable mode imager</td>
			<td>Amersham Biosciences</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

efficient and site-specific antibody labeling by strain-promoted azide-alkyne cycloaddition

Ci sono attualmente molti strumenti chimici a disposizione per introdurre sonde chimiche in proteine ​​per studiare la loro struttura e funzione. Un metodo utile è la coniugazione di proteine ​​geneticamente introducendo un amminoacido non naturale contenente un gruppo funzionale bioorthogonal. Questo rapporto descrive un protocollo dettagliato per site-specific anticorpi coniugazione. Il protocollo comprende dettagli sperimentali per la costituzione genetica di un aminoacido azide contenenti, e la reazione di coniugazione per cicloaddizione azide-alchino strain-promosso (SPAAC). Questa reazione strain-promosso procede per semplice miscelazione delle molecole reagenti a pH fisiologico e temperatura, e non richiede reagenti aggiuntivi come rame (I) ioni e leganti rame-chelanti. Pertanto, questo metodo sarebbe utile per la coniugazione di proteine ​​generale e sviluppo di anticorpi coniugati farmaco (ADC).

<html> In questo studio, un frammento di anticorpo era sito-specifico coniugato con un fluorocromo incorporando un aminoacido azide contenente nel frammento e reagire il frammento di anticorpo mutante con un cyclooctyne teso (Figura 1). HerFab è stato selezionato come il frammento di anticorpo di destinazione in cui AF è stato accolto come un aminoacido azide contenenti. Per scegliere il residuo in HerFab per la sostituzione con AF, la struttura cristallina ai raggi X d...

Watch this Scientific Journal Video about Efficient and Site-specific Antibody Labeling by Strain-promoted Azide-alkyne Cycloaddition at JoVE.com

Efficient and Site-specific Antibody Labeling by Strain-promoted Azide-alkyne Cycloaddition

Here, we present a protocol to site-specifically introduce chemical probes into an antibody fragment by genetically incorporating an azide-containing amino acid, and subsequently coupling the azide with a chemical probe by strain-promoted azide-alkyne cycloaddition (SPAAC).

Efficiente e anticorpi etichettatura per cicloaddizione azide-alchino Strain-promosso site-specific

There are currently many chemical tools available to introduce chemical probes into proteins to study their structure and function. A useful method is ...

Research

JoVE Journal

Biochemistry

Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling

L&#39;identificazione di piccole proteine ​​che legano molecole in un nativo Cellular ambiente da Live-cell Photoaffinity Etichettatura

Identifying the molecular target(s) of small molecules is a challenging but necessary step towards understanding their mechanism of action. While several ...

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Bacterial Inner-membrane Display for Screening a Library of Antibody Fragments

Display interno-membrana batterica per lo screening di una libreria di frammenti di anticorpi

Antibodies engineered for intracellular function must not only have affinity for their target antigen, but must also be soluble and correctly folded in ...

A Facile and Efficient Approach for the Production of Reversible Disulfide Cross-linked Micelles

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Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. ...

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Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the ...

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Enhanced Sample Multiplexing of Tissues Using Combined Precursor Isotopic Labeling and Isobaric Tagging cPILOT

Maggiore Campione Multiplexing dei tessuti utilizzando combinata Precursore isotopica etichettatura e isobarica Tagging (CPilot)

There is an increasing demand to analyze many biological samples for disease understanding and biomarker discovery. Quantitative proteomics strategies ...

Determination of High-affinity Antibody-antigen Binding Kinetics Using Four Biosensor Platforms

Determinazione di alta affinità antigene-anticorpo cinetica del legame con quattro piattaforme biosensore

Label-free optical biosensors are powerful tools in drug discovery for the characterization of biomolecular interactions. In this study, we describe the ...

Analysis of Histone Antibody Specificity with Peptide Microarrays

Analisi della specificità degli anticorpi istonici con microarray di peptide

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The ...

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

Proteoma profonda profilatura di etichettatura isobarico, vasto cromatografia liquida, spettrometria di massa e quantificazione assistita da Software

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) ...

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

Metodo per ripiegamento efficiente e purificazione del chemocettore Ligand Binding Domain

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be ...

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

Isolamento dei Glomeruli e In Vivo di etichettatura delle proteine di superficie delle cellule glomerulari

Proteinuria results from the disruption of the glomerular filter that is composed of the fenestrated endothelium, glomerular basement membrane, and ...