Name: optimizing the genetic incorporation of chemical probes into gpcrs for photo-crosslinking mapping and bioorthogonal chemistry in live mammalian cells
Uploaded: 2018-04-09T14:30:00
Description: Watch this Scientific Journal Video about Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells at JoVE.

यह काम ड्यूश Forschungsgemeinschaft (DFG) द्वारा अनुदान CO822 के तहत स्थापित किया गया है/2-1 (एमी-Noether कार्यक्रम) और CO822/3-1 से आईसी

1. प्रतिदीप्ति-निगमन क्षमता के आधार पर स्क्रीनिंग (चित्रा 1)
<ol><li>Dulbecco के संशोधित ईगल के माध्यम में HEK293 कोशिकाओं को बनाए रखने (DMEM; उच्च ग्लूकोज, 4 मिमी glutamine, पाइरूवेट) 10% के साथ पूरक (वी/वी) भ्रूण गोजातीय ?...

प्रोटोकॉल का वर्णन एक सरल और विश्वसनीय परख के लिए ncaas के शामिल करने के लिए ओर्थोगोनल जोड़े की दक्षता का आकलन प्रोटीन में स्तनधारी कोशिकाओं में व्यक्त की । FACS के आधार पर व्यापक रूप से इस्तेमाल किया परख के ?...

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acryamide/Bisacrylamide 30% (37,5:1)</td>
			<td>Carl Roth</td>
			<td>3029.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium persulfate (APS)</td>
			<td>Carl Roth</td>
			<td>9592.2</td>
			<td></td>
		</tr>
		<tr>
			<td>p-Azidophenylalanine (Azi)</td>
			<td>Bachem</td>
			<td>F-3075.0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Boric acid</td>
			<td>Sigma Aldrich</td>
			<td>B6768</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromphenolblue</td>
			<td>Sigma-Aldrich</td>
			<td>B0126-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumine (BSA)</td>
			<td>Carl Roth</td>
			<td>8076.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbobenzyloxy-L-lysine (Lys(Z))</td>
			<td>NovaBiochem</td>
			<td>8540430100</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyclooctyne-L-lysine (SCOK)</td>
			<td>Sichem</td>
			<td>SC-8000</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Life Technologies</td>
			<td>41966052</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Carl Roth</td>
			<td>A994.2</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Carl Roth</td>
			<td>6908.1</td>
			<td></td>
		</tr>
		<tr>
			<td>enhanced chemiluminescence reagent (ECL)&#160;</td>
			<td>home-made</td>
			<td></td>
			<td>10 mg/l luminol in 0.1 M Tris-HCl pH 8.6 ; 1100 mg/l&#160; p-coumaric acid in DMSO ; 30&#160;% H2O2 (1,000 : 100 : 0.3) [Quelle Laborjounal]</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Carl Roth</td>
			<td>8043.1</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Carl Roth</td>
			<td>3054.1</td>
			<td></td>
		</tr>
		<tr>
			<td>endo-bicyclo[6.1.0]nonyne-L-lysine (BCNK)</td>
			<td>Sichem</td>
			<td>SC-8014</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Thermo Fisher (Gibco)</td>
			<td>10270106</td>
			<td></td>
		</tr>
		<tr>
			<td>FluoroBrite DMEM</td>
			<td>Thermo Fisher (Gibco)</td>
			<td>A1896701</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Carl Roth</td>
			<td>7533.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycin</td>
			<td>Carl Roth</td>
			<td>3908.3</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Carl Roth</td>
			<td>9105.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Sigma Aldrich</td>
			<td>B2261</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Carl Roth</td>
			<td>6781.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000&#160;</td>
			<td>Thermo Fisher</td>
			<td>11668019</td>
			<td></td>
		</tr>
		<tr>
			<td>Luminol</td>
			<td>Applichem</td>
			<td>A2185,0005</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Carl Roth</td>
			<td>0082.3</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Carl Roth</td>
			<td>2189.2</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Carl Roth</td>
			<td>HN00.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Na-Lactate</td>
			<td>Sigma-Aldrich</td>
			<td>71718-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Gr&#252;ssing</td>
			<td>121551000</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma-Aldrich</td>
			<td>P5493-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>p-Coumaric acid</td>
			<td>Sigma-Aldrich</td>
			<td>C9008-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>poly-D-lysine hydrobromide</td>
			<td>Corning</td>
			<td>354210</td>
			<td></td>
		</tr>
		<tr>
			<td>PEI</td>
			<td>Polysciences</td>
			<td>23966</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Thermo Fisher (Gibco)</td>
			<td>11548876 (15140-122)</td>
			<td></td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Carl Roth</td>
			<td>6367.1</td>
			<td></td>
		</tr>
		<tr>
			<td>PNGase F</td>
			<td>NEB</td>
			<td>P0704L</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitor</td>
			<td>Roche</td>
			<td>11873580001</td>
			<td></td>
		</tr>
		<tr>
			<td>PVDF membrane Immobilon-P</td>
			<td>Millipore</td>
			<td>IPVH00010</td>
			<td></td>
		</tr>
		<tr>
			<td>Skim Milk Powder&#160;</td>
			<td>Sigma</td>
			<td>70166</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>Carl Roth</td>
			<td>CN30.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetrazine-Cy3</td>
			<td>Jena Bioscience</td>
			<td>CLK-014-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetramethylethylenediamine (TEMED)</td>
			<td>Carl Roth</td>
			<td>2367.3</td>
			<td></td>
		</tr>
		<tr>
			<td>trans-Cyclooctene-L-lysine (TCO*K)</td>
			<td>Sichem</td>
			<td>SC-8008</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIS</td>
			<td>Sigma-Aldrich</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Carl Roth</td>
			<td>3051.4</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin 2.5%</td>
			<td>Thermo Fisher (Gibco)</td>
			<td>15090046</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Carl Roth</td>
			<td>9127.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Wasserstoffperoxid (30%)</td>
			<td>Merck</td>
			<td>1.07210.0250</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell lines</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HEK293 cells</td>
			<td>German Collection of Microorganisms and Cell Cultures GmbH (DSMZ)</td>
			<td>ACC-305</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK293T cells</td>
			<td>German Collection of Microorganisms and Cell Cultures GmbH (DSMZ)</td>
			<td>ACC-635</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Crosslinker Bio-Link 365 nm</td>
			<td>Bio-Budget Technologies GmbH</td>
			<td>40-BLX-E365</td>
			<td>5 x 8 Watt tubes</td>
		</tr>
		<tr>
			<td>Plate Reader BMG LABTECH FLUOstar Omega&#160;</td>
			<td>BMG LABTECH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmids</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid E2AziRS</td>
			<td>The huminized gene for E2AziRS was synthesized by Geneart (Life Technologies)</td>
			<td></td>
			<td>Plasmid containing 4 tandem copies of the suppressor tRNA Bst-Yam driven by the human U6 promoter and one copy of a humanized gene for the enhanced variant of the Azi-tRNA synthetase (EAziRS) driven by a PGK promoter</td>
		</tr>
		<tr>
			<td>POI-TAG mutant plasmids</td>
			<td></td>
			<td></td>
			<td>Plasmid encoding the POI driven by the CMV promoter, C-terminally fused to the FLAG-tag, bearing a TAG codon at the desired position&#160;</td>
		</tr>
		<tr>
			<td>CRF1R-95TAG-EGFP</td>
			<td></td>
			<td></td>
			<td>Cloned in the MCS of pcDNA3.1</td>
		</tr>
		<tr>
			<td>HA-PTH1R-79TAG-CFP</td>
			<td></td>
			<td></td>
			<td>Cloned in the MCS of pcDNA3.1</td>
		</tr>
		<tr>
			<td>Arrestin3-FLAG</td>
			<td>Synthesized by Genart (Life Technologies)</td>
			<td></td>
			<td>Cloned in the MCS of pcDNA3.1</td>
		</tr>
		<tr>
			<td>Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-FLAG-HRP M2 antibody conjugate&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>A8592&#160;</td>
			<td>monoclonal, produced in mouse clone M2</td>
		</tr>
		<tr>
			<td>Goat-anti-rabbit-HRP antibody</td>
			<td>Santa Cruz</td>
			<td>sc-2004</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit-anti-CRF antibody</td>
			<td>home-made</td>
			<td>PBL #rC69</td>
			<td>polyclonal [Turnbull]</td>
		</tr>
		<tr>
			<td>Rabbit-anti-Ucn1 antibody</td>
			<td>home-made</td>
			<td>PBL #5779</td>
			<td>polyclonal [Turnbull]</td>
		</tr>
	</tbody>
</table>

optimizing the genetic incorporation of chemical probes into gpcrs for photo-crosslinking mapping and bioorthogonal chemistry in live mammalian cells

गैर के आनुवंशिक शामिल-विहित अमीनो एसिड (ncaas) एंबर रोक codon दमन के माध्यम से एक शक्तिशाली तकनीक को सीधे जीवित कोशिका में प्रोटीन पर कृत्रिम जांच और प्रतिक्रियाशील moieties स्थापित है । प्रत्येक ncAA एक समर्पित ओर्थोगोनल दमन-tRNA/एमिनो-acyl-tRNA-synthetase (AARS) जोड़ी है कि मेजबान जीव में आयात किया जाता है द्वारा शामिल है । विभिंन ncaas के निगमन दक्षता बहुत अलग कर सकते हैं, और कुछ मामलों में असंतोषजनक है । ओर्थोगोनल जोड़े या तो AARS या tRNA जोड़ तोड़ से सुधार किया जा सकता है । हालांकि, tRNA या बड़े पुस्तकालयों और मृत/जिंदा चयन विधियों का उपयोग AARS के विकास का निर्देश स्तनधारी कोशिकाओं में व्यवहार्य नहीं हैं । इधर, स्तनधारी कोशिकाओं में ओर्थोगोनल जोड़े की कुशलता का मूल्यांकन करने के लिए एक सतही और दमदार प्रतिदीप्ति आधारित परख प्रस्तुत की गई है. परख एक उदारवादी प्रयास के साथ AARS/tRNA वेरिएंट के सैकड़ों को दसियों स्क्रीनिंग की अनुमति देता है और एक उचित समय के भीतर । इस परख का उपयोग करने के लिए नए tRNAs कि काफी pyrrolysine ओर्थोगोनल प्रणाली की क्षमता में सुधार उत्पंन करने के साथ वर्णित है, जी के अध्ययन के लिए ncaas के आवेदन के साथ प्रोटीन युग्मित रिसेप्टर्स (GPCRs), जो ncaa के लिए वस्तुओं को चुनौती दे रहे हैं mutagenesis । पहले, व्यवस्थित एक रिसेप्टर के extracellular सतह भर में एक तस्वीर crosslinking ncAA शामिल करके, बरकरार रिसेप्टर पर अलग लाइगैंडों के बंधन साइटों सीधे लाइव सेल में मैप कर रहे हैं. दूसरा, एक GPCR, ultrafast उत्प्रेरक-मुक्त रिसेप्टर एक फ्लोरोसेंट डाई के साथ लेबलिंग में अंतिम पीढ़ी के ncaa को शामिल करके प्रदर्शन किया है, जो bioorthogonal तनाव-पदोंनत व्युत्क्रम Diels Alder cycloaddition (SPIEDAC) जी सेल पर बढ़ावा दिया । के रूप में ncaas आम तौर पर किसी भी प्रोटीन के लिए स्वतंत्र रूप से अपने आकार पर लागू किया जा सकता है, विधि आवेदनों की एक संख्या के लिए सामांय ब्याज की है । इसके अलावा, ncAA निगमन किसी विशेष उपकरण की आवश्यकता नहीं है और आसानी से मानक जैव रसायन प्रयोगशालाओं में प्रदर्शन किया ।

प्रतिदीप्ति परख की रूपरेखा चित्रा 1में दिखाया गया है । परख तीन आवेदनों में कार्यरत है । फर्स्ट प्लेस में Pyl ओर्थोगोनल पेयर द्वारा Lys (बीओसी) के लिए tRNA वेरिएंट की संख्या दिखलाई जाती ?...

Watch this Scientific Journal Video about Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells at JoVE.

Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells

एक सतही प्रतिदीप्ति परख अमीनो की दक्षता का मूल्यांकन करने के लिए प्रस्तुत किया जाता है-acyl-tRNA-synthetase/tRNA जोड़े शामिल गैर विहित अमीनो-एसिड (ncaas) प्रोटीन में स्तनधारी कोशिकाओं में व्यक्त की । जी का अध्ययन करने के लिए ncaas के आवेदन प्रोटीन युग्मित रिसेप्टर्स (GPCRs) फोटो crosslinking बाइंडिंग साइटों की मानचित्रण और bioorthogonal GPCR रहते कोशिकाओं पर लेबलिंग सहित वर्णित है ।

फोटो के लिए GPCRs में रासायनिक जांच के आनुवंशिक निगमन-crosslinking मानचित्रण और लाइव स्तनधारी कोशिकाओं में Bioorthogonal रसायन विज्ञान का अनुकूलन

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and ...

The overall goal of this protocol is to enhance the incorporation rate of non-canonical amino acids in mammalian cells to tackle biological questions in live cell settings including mapping of protein-protein interaction surfaces, and bioorthogonal labeling of surface receptors. Using amber codon suppression, different non-canonical amino acids are incorporated with different efficiencies. We established fluorescence assay to easily compare non-canonical amino acids incorporation rates in mammalian cells within a short time.The systematic incorporation of photo-crosslinking, non-canonical amino acids through our GPCR surfaces allows mapping of ligand binding sites and provides precious information of the intake receptor directly from the life cell. By incorporating last generation, non-canonical amino acids for bioorthogonal chemistry, we achieve site-specific, super fast, catalyst free labeling of GPCRs with small, bright fluorophores for high-end imaging applications in live mammalian cells. First, seed 600, 000 HEK 293 cells in two milliliters of complete growth medium per well in two six-well plates.Prepare as many wells as the number of samples and two additional wells for the wild type EGFP and a mock transfected sample respectively. One hour prior to transfection, add the appropriate amount of freshly prepared non-canonical amino acid stock solution to all wells for a final amino acid concentration of 0.25 to 0.5 millimolar. To transfect the cells, mix one microgram of plasmid DNA and coding for the synthetase t-RNA pair to be tested with one microgram of reporter plasmid DNA in a microcentrifuge tube.In separate tubes, prepare an identical transfection using the EGFP wild type reference and a mock transfection. Next, add 100 microliters of lactate buffered saline to each tube containing the DNA. After briefly mixing, add six microliters of one microgram per microliter PEI and lactate buffered saline to each tube.Vortex immediately, and then incubate at room temperature for 10 to 15 minutes. Transfer 400 microliters of cell medium from each well to the DNA PEI mixture to neutralize the pH. Then, dribble the DNA mixture onto the cells.Harvest the cells 48 hours post-transfection by aspirating the medium and rinsing the cells once with two milliliters of pre-warmed PBS. Add 800 microliters of PBS supplemented with 0.5 millimolar EDTA, and incubate for 20 minutes at 37 degrees Celsius. After incubation, detach and suspend the cells by pipetting up and down.Next, transfer the cell suspension into microcentrifuge tubes containing 200 microliters of PBS supplemented with five millimolar magnesium chloride. Centrifuge the samples for two minutes at 800 times gravity. When finished, discard the supernatant.Now, add 100 microliters of Tris lysis buffer to the cell pellets and incubate on ice for 30 minutes. To facilitate lysis, vortex every five minutes. Spin down the cell debris for 10 minutes at four degrees Celsius and 14, 000 times gravity.Transfer 90 microliters of the supernatant into black 96-well plates. Then, measure EGFP and mCherry fluorescence using a plate reader equipped with the fluorescence module. Seed 500, 000 293 t-cells in two milliliters of complete growth medium per well in two six-well plates.For each position to be screened, prepare one well per ligand, plus one well for the binding control. One hour prior to transfection, add the photo-crosslinker, p-azidophenylalanine, or Azi, to all wells to a final concentration of 0.5 millimolar. Transfect a total amount of two micrograms of DNA per well using one microgram of plasmid and coding for the flag tagged GPCR bearing a tag codon at the desired position, and one microgram of the plasmid and coding for the orthogonal pair dedicated to Azi.At 48 hours post-transfection, prepare a 1, 000x ligand stock solution by dissolving the peptide ligand at a concentration of 100 micromolar in DMSO. Dilute the ligand stock solution one to 1, 000 in binding buffer. Replace the cell medium with one milliliter of the ligand solution and incubate the samples for 10 minutes at room temperature.Following incubation, irradiate the cells for 20 minutes in a UV cross-linker at 365 nanometers and five centimeter distance to the cells. When finished, detach the cells by pipetting and transfer them into a microcentrifuge tube. Pellet the cells for three minutes at 800 times gravity.After discarding the supernatant, resuspend the cell pellets in 50 microliters of HEPES dissociation buffer, or HDB, containing a cocktail of protease inhibitors. Then, flash freeze the cells in liquid nitrogen. Next, thaw the cells in a water bath at 37 degrees Celsius for 30 to 45 seconds, and then vortex briefly.Pellet the membranes for 10 minutes at four degrees Celsius and 2500 times gravity. When finished, discard the supernatant which contains the bulk of cytosolic proteins. Thoroughly resuspend the pellets in 50 microliters of HEPES lysis buffer using a pipet.Then, lyse the cells for 30 minutes on ice, vortexing every five minutes. Following this, spin down the cell debris for 10 minutes at four degrees Celsius and 14, 000 times gravity. Immediately transfer each supernatant to a fresh reaction tube for western blot analysis.When the GPCR is glycosylated and faint or smeared bands are a problem in western blot analysis, deglycosylate the samples with PNGase F to increase signal intensity and sharpen the bands. Then, resolve the samples on standard SDS page and blot transfer proteins to a PVDF membrane. After blocking the membrane, probe it with an anti-ligand antibody to detect the occurrence of cross-linking.To detect the expression level of the Azi-GPCR, probe the membrane with a commercial anti-flag antibody. After washing the membrane, soak it in enhanced chemiluminescence solution, and visualize the signals in the dark. Seed 100, 000 HEK 293 t-cells in 600 microliters of dye-free, complete DMEM per well of a microscopy slide equipped with four wells.One hour prior to transfection, prepare a fresh 100 millimolar stock solution of TCOK and 0.2 molar sodium hydroxide and 15%DMSO. Mix three microliters of the TCOK stock solution with 12 microliters of one molar, pH 7.4 HEPES buffer. Gently add the solution to each well for a final TCOK concentration of 0.5 millimolar.In a microcentrifuge tube, dilute 200 nanograms of the plasmid and coding for the receptor containing the amber codon and 200 nanograms of the plasmid and coding for the orthogonal pair in 50 microliters of medium. Dilute 1.25 microliters of lipofectamine 2, 000 in 50 microliters of dye-free, serum-free, and antibiotic-free medium, and add the solution to the DNA solution. Vortex immediately and incubate for five to 10 minutes at room temperature.Then, add the DNA lipid complexes to the cells. At 24 hours post-transfection, transfer 100 microliters of medium from each well into a 1.5 milliliter reaction tube. Add 1.8 microliters of an orange fluorescent dye tetrazine stock solution, and 0.3 microliters of a DNA staining dye stock solution.Transfer the medium containing the dyes back to the well and incubate for five minutes at 37 degrees Celsius. Aspirate the medium, and gently rinse the cells twice with PBS to remove the excess dye. Then, add 600 microliters of pre-heated, complete dye-free growth medium to the cells.Now, visualize the labeled receptors under 63x magnification using filters appropriate for GFP orange fluorescent dye and DNA-staining dye. After adding peptide agonist stock solution to the cells, observe the internalization under the microscope using the aforementioned filters and take pictures after the clearly detectable occurrence of internalization. Different tRNA's give different suppression efficiency, which is clearly reflected in the amount of measured fluorescence.Azi incorporation into CRF1R was highly enhanced when using the codon-optimized Azi-tRNA synthetase gene compared to the native gene, showing that even a moderate improvement for a soluble protein can have a greater impact on the expression level of more challenging membrane proteins. The optimized system for Azi incorporation, including the humanized E2 Azi RS gene was deployed to map the binding pocket of the CRF1R and to find binding paths of five ligands. A band at the correct size of the crosslinking product in the western blot reveals that the position lies in the proximity of the ligand within the ligand receptor complex.Multiple crosslinking hits with all ligands tested revealed distinct binding patterns for the peptide Agonists and Antagonists. Both fluorescence and western blot data suggests that TCOK is the non-canonical amino acids for bioorthogonal chemistry that gets incorporated with the highest efficiency of the AF variant of the paralysine tRNA synthetase. TCOK was incorporated into a CRF1R EGFP fusion protein and enabled installing a small, bright fluorophore on the receptor.After adding a peptide Agaonist, fluorescent compartments were observed throughout the cytosol, revealing the physiological process of GPCR internalization. To do a fluorescence assay, we presented here is a simple and effective tool to evaluate and optimize the efficiency of a synthetase TI and API in mammalian cells. High incorporation rates are crucial for applying non-canonical amino acids to investigate challenging biological questions.Highly efficient incorporation of crosslinking non-canonical amino acids enables extensive surface mapping of virtually any protein, including membrane receptors or low-banded proteins. The method reveals the topology of interaction interfaces which can be further analyzed to obtain accurate molecular models. High incorporation efficiency of last generation, non-canonical amino acids for bioorthogonal chemistry allows us to equip GPCRs with small, bright fluorophores, thus leading the way towards the study of GPCRs structural dynamics in live cells via advanced microscopy techniques.

Research

JoVE Journal

Chemistry

Isolation and Chemical Characterization of Lipid A from Gram-negative Bacteria

ग्राम नकारात्मक बैक्टीरिया से अलगाव और लिपिड एक के रासायनिक लक्षण वर्णन

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS ...

A Simple and Rapid Protocol for Measuring Neutral Lipids in Algal Cells Using Fluorescence

प्रतिदीप्ति का उपयोग algal कोशिकाओं में तटस्थ लिपिड मापने के लिए एक सरल और तेजी से प्रोटोकॉल

Algae are considered excellent candidates for renewable fuel sources due to their natural lipid storage capabilities. Robust monitoring of algal ...

Preparing Adherent Cells for X-ray Fluorescence Imaging by Chemical Fixation

रासायनिक फिक्सेशन द्वारा एक्स-रे प्रतिदीप्ति इमेजिंग के लिए पक्षपाती कोशिकाओं की तैयारी

X-ray fluorescence imaging allows us to non-destructively measure the spatial distribution and concentration of multiple elements simultaneously over ...

Chemical Vapor Deposition of an Organic Magnet, Vanadium Tetracyanoethylene

एक कार्बनिक चुंबक की रासायनिक वाष्प जमाव, Vanadium Tetracyanoethylene

Recent progress in the field of organic materials has yielded devices such as organic light emitting diodes (OLEDs) which have advantages not found in ...

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical (Methylation) and Physical (Mass Spectrometry, Nuclear Magnetic Resonance) Techniques

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical Methylation and Physical Mass Spectrometry, Nuclear Magnetic Resonance Techniques

संयंत्र दीवार Heteroxylans का अनुक्रमण Enzymic, रासायनिक (मेथिलिकरण) और शारीरिक (मास स्पेक्ट्रोमेट्री, परमाणु चुंबकीय अनुनाद) तकनीकों का प्रयोग

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of ...

Immobilization of Multi-biocatalysts in Alginate Beads for Cofactor Regeneration and Improved Reusability

सहायक कारक के उत्थान और बेहतर reusability के लिए Alginate मोती में मल्टी biocatalysts का स्थिरीकरण

We have recently developed a simple, reusable and coupled whole-cell biocatalytic system with the capability of cofactor regeneration and biocatalyst ...

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

प्रोटीन Bioconjugates के संश्लेषण<em&gt; के माध्यम से</em&gt; सिस्टीन maleimide कैमिस्ट्री

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

The Preparation and Properties of Thermo-reversibly Cross-linked Rubber Via Diels-Alder Chemistry

तैयारी और थर्मो-reversibly क्रॉस से जुड़े रबड़ के माध्यम से Diels-Alder रसायन विज्ञान के गुण

A method for using Diels Alder thermo-reversible chemistry as cross-linking tool for rubber products is demonstrated. In this work, a commercial ...

Insights into the Interactions of Amino Acids and Peptides with Inorganic Materials Using Single-Molecule Force Spectroscopy

एमिनो एसिड और पेप्टाइड्स अकार्बनिक सामग्री के साथ बातचीत में इनसाइट्स एकल अणु बल स्पेक्ट्रोस्कोपी का उपयोग

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals ...

Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry

Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry

तैयारी और का मूल्यांकन<sup&gt; 99m</sup&gt; टीसी लेबल प्री-लक्ष्य Bioorthogonal रसायन विज्ञान का उपयोग के लिए Tridentate chelates

Pre-targeting combined with bioorthogonal chemistry is emerging as an effective way to create new radiopharmaceuticals. Of the methods available, the ...