Name: native cell membrane nanoparticles system for membrane protein-protein interaction analysis
Uploaded: 2020-07-16T13:30:00
Description: Watch this Scientific Journal Video about Native Cell Membrane Nanoparticles System for Membrane Protein-Protein Interaction Analysis at JoVE.com

इस शोध को वीसीयू स्टार्टअप फंड (वाईजी को) और नेशनल इंस्टीट्यूट ऑफ हेल्थ के नेशनल इंस्टीट्यूट ऑफ जनरल मेडिकल साइंसेज द्वारा पुरस्कार संख्या R01GM132329 (वाईजी को) के तहत समर्थित किया गया था । सामग्री पूरी तरह से लेखकों की जिम्मेदारी है और जरूरी नहीं कि स्वास्थ्य के राष्ट्रीय संस्थानों के आधिकारिक विचारों का प्रतिनिधित्व करता है । हम वीडियो रिकॉर्डिंग के लिए उनके उदार समर्थन के लिए मोंटसराट सैमसो और केविन मैकरॉबर्ट्स को धन्यवाद देते हैं।

1. प्रोटीन अभिव्यक्ति
<ol><li>BL21 (DE3) pLysS कोशिकाओं के साथ प्लाज्मिड के लिए विशिष्ट एंटीबायोटिक दवाओं के साथ भयानक शोरबा (टीबी) मीडिया के 15 एमएल टीका 250 आरपीएम पर मिलाते हुए 37 डिग्री सेल्सियस पर रात भर 50 एमएल ट्यूब म...

<ol>
	<li>Berggard, T., Linse, S., James, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+the+detection+and+analysis+of+protein-protein+interactions.">Methods for the detection and analysis of protein-protein interactions.</a> Proteomics. 7 (16), 2833-2842 (2007).</li><li>Huang, H., Bader, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precision+and+recall+estimates+for+two-hybrid+screens.">Precision and recall estimates for two-hybrid screens.</a> Bioinformatics. 25 (3), 372-378 (2009).</li><li>Serebriiskii, I. G., Golemis, 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=Two-hybrid+system+and+false+positives.+Approaches+to+detection+and+elimination.">Two-hybrid system and false positives. Approaches to detection and elimination.</a> Methods in Molecular Biology. 177, 123-134 (2001).</li><li>Gingras, A. C., Gstaiger, M., Raught, B., Aebersold, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+protein+complexes+using+mass+spectrometry.">Analysis of protein complexes using mass spectrometry.</a> Nature Reviews in Molecular Cell Biology. 8 (8), 645-654 (2007).</li><li>Ngounou Wetie, A. G., 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=Investigation+of+stable+and+transient+protein-protein+interactions:+Past,+present,+and+future.">Investigation of stable and transient protein-protein interactions: Past, present, and future.</a> Proteomics. 13 (3-4), 538-557 (2013).</li><li>Schaufele, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maximizing+the+quantitative+accuracy+and+reproducibility+of+Forster+resonance+energy+transfer+measurement+for+screening+by+high+throughput+widefield+microscopy.">Maximizing the quantitative accuracy and reproducibility of Forster resonance energy transfer measurement for screening by high throughput widefield microscopy.</a> Methods. 66 (2), 188-199 (2014).</li><li>Berney, C., Danuser, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FRET+or+no+FRET:+a+quantitative+comparison.">FRET or no FRET: a quantitative comparison.</a> Biophysical Journal. 84 (6), 3992-4010 (2003).</li><li>Almen, M. S., Nordstrom, K. J., Fredriksson, R., Schioth, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+the+human+membrane+proteome:+a+majority+of+the+human+membrane+proteins+can+be+classified+according+to+function+and+evolutionary+origin.">Mapping the human membrane proteome: a majority of the human membrane proteins can be classified according to function and evolutionary origin.</a> BMC Biology. 7, 50 (2009).</li><li>Overington, J. P., Al-Lazikani, B., Hopkins, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+many+drug+targets+are+there.">How many drug targets are there.</a> Nature Reviews in Drug Discovery. 5 (12), 993-996 (2006).</li><li>Berman, H. M., 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=The+Protein+Data+Bank.">The Protein Data Bank.</a> Nucleic Acids Research. 28 (1), 235-242 (2000).</li><li>Overduin, M., Esmaili, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Memtein:+The+fundamental+unit+of+membrane-protein+structure+and+function.">Memtein: The fundamental unit of membrane-protein structure and function.</a> Chemistry and Physics of Lipids. 218, 73-84 (2019).</li><li>Qiu, 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=Structure+and+activity+of+lipid+bilayer+within+a+membrane-protein+transporter.">Structure and activity of lipid bilayer within a membrane-protein transporter.</a> Proceedings of the National Academy of Sciences U. S. A. 115 (51), 12985-12990 (2018).</li><li>Knowles, T. 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=Membrane+proteins+solubilized+intact+in+lipid+containing+nanoparticles+bounded+by+styrene+maleic+acid+copolymer.">Membrane proteins solubilized intact in lipid containing nanoparticles bounded by styrene maleic acid copolymer.</a> Journal of the American Chemical Society. 131 (22), 7484-7485 (2009).</li><li>Xue, M., Cheng, L., Faustino, I., Guo, W., Marrink, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Mechanism+of+Lipid+Nanodisk+Formation+by+Styrene-Maleic+Acid+Copolymers.">Molecular Mechanism of Lipid Nanodisk Formation by Styrene-Maleic Acid Copolymers.</a> Biophysical Journal. 115 (3), 494-502 (2018).</li><li>Parmar, M., 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=Using+a+SMALP+platform+to+determine+a+sub-nm+single+particle+cryo-EM+membrane+protein+structure.">Using a SMALP platform to determine a sub-nm single particle cryo-EM membrane protein structure.</a> Biochimica and Biophysica Acta Biomembrames. 1860 (2), 378-383 (2018).</li><li>Yu, L., Lu, W., Wei, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AcrB+trimer+stability+and+efflux+activity,+insight+from+mutagenesis+studies.">AcrB trimer stability and efflux activity, insight from mutagenesis studies.</a> PLoS One. 6 (12), 28390 (2011).</li><li>Broussard, J. A., Rappaz, B., Webb, D. J., Brown, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+resonance+energy+transfer+microscopy+as+demonstrated+by+measuring+the+activation+of+the+serine/threonine+kinase+Akt.">Fluorescence resonance energy transfer microscopy as demonstrated by measuring the activation of the serine/threonine kinase Akt.</a> Nature Protocols. 8 (2), 265-281 (2013).</li><li>Ma, L., Yang, F., Zheng, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+fluorescence+resonance+energy+transfer+in+protein+studies.">Application of fluorescence resonance energy transfer in protein studies.</a> Journal of Molecular Structure. 1077, 87-100 (2014).</li><li>Sachl, R., Humpolickova, J., Stefl, M., Johansson, L. B., Hof, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limitations+of+electronic+energy+transfer+in+the+determination+of+lipid+nanodomain+sizes.">Limitations of electronic energy transfer in the determination of lipid nanodomain sizes.</a> Biophysical Journal. 101 (11), 60-62 (2011).</li><li>Woehler, A., Wlodarczyk, J., Neher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal/noise+analysis+of+FRET-based+sensors.">Signal/noise analysis of FRET-based sensors.</a> Biophysical Journal. 99 (7), 2344-2354 (2010).</li><li>Wang, 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=Comparison+of+in+vitro+and+in+vivo+oligomeric+states+of+a+wild+type+and+mutant+trimeric+inner+membrane+multidrug+transporter.">Comparison of in vitro and in vivo oligomeric states of a wild type and mutant trimeric inner membrane multidrug transporter.</a> Biochemical and Biophysical Reports. 16, 122-129 (2018).</li><li>Scarff, C. A., Fuller, M. J. G., Thompson, R. F., Iadaza, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variations+on+Negative+Stain+Electron+Microscopy+Methods:+Tools+for+Tackling+Challenging+Systems.">Variations on Negative Stain Electron Microscopy Methods: Tools for Tackling Challenging Systems.</a> Journal of Visualized Experiments. (132), e57199 (2018).</li><li>Lu, W., Zhong, M., Wei, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Folding+of+AcrB+Subunit+Precedes+Trimerization.">Folding of AcrB Subunit Precedes Trimerization.</a> Journal of Molecular Biology. 411 (1), 264-274 (2011).</li><li>Lebon, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Structure and Function of GPCRs. , 229-230 (2019).</li><li>Rames, M., Yu, Y., Ren, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+negative+staining:+a+high-throughput+protocol+for+examining+small+and+asymmetric+protein+structure+by+electron+microscopy.">Optimized negative staining: a high-throughput protocol for examining small and asymmetric protein structure by electron microscopy.</a> Journal of Visualized Experiments. (90), e51087 (2014).</li><li>Lee, S. C., 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+method+for+detergent-free+isolation+of+membrane+proteins+in+their+local+lipid+environment.">A method for detergent-free isolation of membrane proteins in their local lipid environment.</a> Nature Protocols. 11 (7), 1149-1162 (2016).</li><li>Hall, S. C. 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=An+acid-compatible+co-polymer+for+the+solubilization+of+membranes+and+proteins+into+lipid+bilayer-containing+nanoparticles.">An acid-compatible co-polymer for the solubilization of membranes and proteins into lipid bilayer-containing nanoparticles.</a> Nanoscale. 10 (22), 10609-10619 (2018).</li><li>Oluwole, A. O., 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=Solubilization+of+Membrane+Proteins+into+Functional+Lipid-Bilayer+Nanodiscs+Using+a+Diisobutylene/Maleic+Acid+Copolymer.">Solubilization of Membrane Proteins into Functional Lipid-Bilayer Nanodiscs Using a Diisobutylene/Maleic Acid Copolymer.</a> Angewandte Chemie International Edition England. 56 (7), 1919-1924 (2017).</li></ol>

झिल्ली प्रोटीन की संरचना और कार्य की अखंडता के लिए प्रोटीन-प्रोटीन इंटरैक्शन महत्वपूर्ण हैं। प्रोटीन-प्रोटीन इंटरैक्शन की जांच के लिए कई दृष्टिकोण विकसित किए गए हैं। घुलनशील प्रोटीन की तुलना में, झि...

<table>
	<tbody>
		<tr>
			<td>Chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>30% Acrylamide/BIS SOL (37.5:1)</td>
			<td>Bio-Rad</td>
			<td>161-0158</td>
			<td></td>
		</tr>
		<tr>
			<td>4x Laemmli Sample Buffer (Loading Buffer)</td>
			<td>Bio-Rad</td>
			<td>1610747</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Acid Glacial</td>
			<td>ThermoFisher Scientific</td>
			<td>A38S-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Persulfate (APS)</td>
			<td>Bio-Rad</td>
			<td>161-0700</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>Goldbio</td>
			<td>C-105-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Coomassie Brilliant Blue R-250 protein stain powder</td>
			<td>Bio-Rad</td>
			<td>161-0400</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT (Dithiothreitol) (&#62; 99% pure) Protease free</td>
			<td>Goldbio</td>
			<td>DTT10</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>ThermoFisher Scientific</td>
			<td>G33-4</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>ThermoFisher Scientific</td>
			<td>BP310-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Affymetrix</td>
			<td>17525 1 KG</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Goldbio</td>
			<td>I2481C100</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>Goldbio</td>
			<td>K-120-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride hexahydrate</td>
			<td>ThermoFisher Scientific</td>
			<td>AA3622636</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>ThermoFisher Scientific</td>
			<td>A412-4</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N-dimethylethylenediamine (EDTA)</td>
			<td>Merck</td>
			<td>8.03779.0100</td>
			<td></td>
		</tr>
		<tr>
			<td>NCMNS-P5-2</td>
			<td>Not commercially available yet</td>
			<td>Submit request for obtaining to corresponding author</td>
			<td></td>
		</tr>
		<tr>
			<td>Precision Plus Protein Dual Color Standard</td>
			<td>Bio-Rad</td>
			<td>161-0374</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS (Sodium Dodecyl Sulfate)</td>
			<td>Bio-Rad</td>
			<td>161-0301</td>
			<td></td>
		</tr>
		<tr>
			<td>SMA2000</td>
			<td>Cray Valley</td>
			<td>Submit request for obtaining to corresponding author</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>ThermoFisher Scientific</td>
			<td>S271-10</td>
			<td></td>
		</tr>
		<tr>
			<td>TCEP-HCl</td>
			<td>Goldbio</td>
			<td>TCEP25</td>
			<td></td>
		</tr>
		<tr>
			<td>TEMED</td>
			<td>Bio-Rad</td>
			<td>161-0800</td>
			<td></td>
		</tr>
		<tr>
			<td>Terrific Broth Media</td>
			<td>Affymetrix</td>
			<td>75856 1 KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Bio-Rad</td>
			<td>161-0719</td>
			<td></td>
		</tr>
		<tr>
			<td>Uranyl Acetate</td>
			<td>Ambinter</td>
			<td>Amb22348393</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Avanti J-26S XPI</td>
			<td>Beckman Coulter</td>
			<td>B14538</td>
			<td></td>
		</tr>
		<tr>
			<td>Avanti JXN-30</td>
			<td>Beckman Coulter</td>
			<td>B34193</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon Electron Microscope Grids (10 nm)</td>
			<td>Electron Microscopy Sciences</td>
			<td>CF300-Cu-TH</td>
			<td></td>
		</tr>
		<tr>
			<td>Con-Torque Tissue Homogenizer</td>
			<td>Eberbach</td>
			<td>E7265</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning LSE Mini Microcentrifuge</td>
			<td>ThermoFisher Scientific</td>
			<td>07-203-954</td>
			<td></td>
		</tr>
		<tr>
			<td>EmulsiFlex-C3</td>
			<td>Avestin</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fraction Collector F9-R</td>
			<td>GE Healthcare Life Sciences</td>
			<td>29003875</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-PROTEAN Tetra Vertical Electrophoresis Cell</td>
			<td>Bio-Rad</td>
			<td>165-8004</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop 2000 Spectrophotometer</td>
			<td>ThermoFisher Scientific</td>
			<td>ND-2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Optima L-90K Ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>PN LL-IM-12AB</td>
			<td></td>
		</tr>
		<tr>
			<td>PELCO easiGlow Glow Discharge Cleaning System</td>
			<td>Ted Pella</td>
			<td>91000S-230</td>
			<td></td>
		</tr>
		<tr>
			<td>Potter-Elvehjem Safe Grind Tissue Grinder</td>
			<td>Wheaton</td>
			<td>358013</td>
			<td></td>
		</tr>
		<tr>
			<td>PowerPac Basic Power Supply</td>
			<td>Bio-Rad</td>
			<td>164-5050</td>
			<td></td>
		</tr>
		<tr>
			<td>Razel R99-E Variable Speed Syringe Pump</td>
			<td>Razel Scientific Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Superdex 200 Increase 10/300 GL</td>
			<td>GE Healthcare Life Sciences</td>
			<td>28990944</td>
			<td></td>
		</tr>
		<tr>
			<td>Tecnai F20 200kV</td>
			<td>FEI</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Type 70 Ti Fixed-Angle Rotor</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>General Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 ml Microcentrifuge Tubes</td>
			<td>ThermoFisher Scientific</td>
			<td>05-408-129</td>
			<td></td>
		</tr>
		<tr>
			<td>4 ml Amicon Ultra-4 30 kDa</td>
			<td>Millipore Sigma</td>
			<td>UFC803024</td>
			<td></td>
		</tr>
		<tr>
			<td>AKTA pure 25 L1 FPLC</td>
			<td>GE Healthcare Life Sciences</td>
			<td>29018225</td>
			<td></td>
		</tr>
		<tr>
			<td>BL21(DE3)pLysS Cells</td>
			<td>ThermoFisher Scientific</td>
			<td>C606003</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 50 ml Conical Centrifuge Tube</td>
			<td>ThermoFisher Scientific</td>
			<td>14-959-49A</td>
			<td></td>
		</tr>
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			<td>HisTrap HP 5 ml Column</td>
			<td>GE Healthcare Life Sciences</td>
			<td>17524802</td>
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			<td>pET-24a</td>
			<td>EMD Biosciences</td>
			<td>69749-3</td>
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native cell membrane nanoparticles system for membrane protein-protein interaction analysis

कोशिका झिल्ली प्रणालियों में प्रोटीन-प्रोटीन इंटरैक्शन जैविक प्रक्रियाओं की एक विस्तृत श्रृंखला में महत्वपूर्ण भूमिका निभाते हैं- सेल-टू-सेल इंटरैक्शन से लेकर संकेत लेनदेन तक; जैविक प्रतिक्रिया के लिए पर्यावरण संकेतों को संवेदन से; मेटाबॉलिक रेगुलेशन से लेकर विकासात्मक नियंत्रण तक। झिल्ली प्रोटीन परिसरों के आणविक तंत्र को समझने और इन प्रोटीनों को मिलाना करने के लिए अत्यधिक विशिष्ट अणुओं के डिजाइन के लिए प्रोटीन-प्रोटीन इंटरैक्शन की सटीक संरचनात्मक जानकारी महत्वपूर्ण है। प्रोटीन-प्रोटीन इंटरैक्शन का पता लगाने और विश्लेषण के लिए वीवो और इन विट्रो दृष्टिकोणों में कई विकसित किए गए हैं। उनमें से संरचनात्मक जीव विज्ञान दृष्टिकोण अद्वितीय है कि यह परमाणु स्तर पर प्रोटीन प्रोटीन बातचीत की प्रत्यक्ष संरचनात्मक जानकारी प्रदान कर सकता है । हालांकि, वर्तमान झिल्ली प्रोटीन संरचनात्मक जीव विज्ञान अभी भी काफी हद तक डिटर्जेंट आधारित तरीकों तक ही सीमित है । डिटर्जेंट आधारित तरीकों की प्रमुख खामी यह है कि वे अक्सर डिटर्जेंट अणुओं द्वारा अपने मूल लिपिड बाइलेयर पर्यावरण को हटा दिए जाने के बाद झिल्ली प्रोटीन परिसरों को अलग या विकृत कर देते हैं। हम झिल्ली प्रोटीन संरचनात्मक जीव विज्ञान के लिए एक देशी कोशिका झिल्ली नैनोपार्टिकल प्रणाली विकसित कर रहे हैं। यहां, हम एसीआरबी की अल्पाधिकारी स्थिति के एक मामले के अध्ययन के साथ कोशिका झिल्ली पर प्रोटीन-प्रोटीन इंटरैक्शन के विश्लेषण में इस प्रणाली के उपयोग को प्रदर्शित करते हैं।

यहां प्रस्तुत प्रक्रियाओं का उपयोग करते हुए ई कोलाई एसीआरबी वाइल्ड टाइप और ई कोलाई उत्परिवर्ती एसीआरबी-पी223जी के नमूनों को शुद्ध किया गया । इसके बाद नमूनों को कार्बन निगेटिव दाग इलेक्ट्रॉन माइ?...

Watch this Scientific Journal Video about Native Cell Membrane Nanoparticles System for Membrane Protein-Protein Interaction Analysis at JoVE.com

Native Cell Membrane Nanoparticles System for Membrane Protein-Protein Interaction Analysis

यहां प्रस्तुत झिल्ली प्रोटीन की अल्पाधिकारी स्थिति के निर्धारण के लिए एक प्रोटोकॉल है जो इलेक्ट्रॉन माइक्रोस्कोपी के साथ मिलकर एक देशी कोशिका झिल्ली नैनोपार्टिकल सिस्टम का उपयोग करता है।

झिल्ली प्रोटीन-प्रोटीन इंटरैक्शन विश्लेषण के लिए देशी सेल झिल्ली नैनोकण प्रणाली

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal ...

This protocol will help researchers more accurately determine the native oligomeric state of membrane proteins by utilizing the native cell membrane nanoparticle system in conjunction with electron microscopy. The advantage of this technique is that it provides accurate structural data of membrane proteins in a native cell membrane-like environment, and it can be utilized for future high-resolution structure determination. Demonstrating the procedure will be Kyle Kroeck, a postdoctoral fellow from my laboratory.To prepare native cell membrane nanoparticles, resuspend one gram of the membrane pellet of interest in 10 milliliters of NCMN Buffer A.Use a glass Dounce homogenizer to homogenize the resuspended cell membrane sample at 20 degrees Celsius and transfer the suspended sample to a 50 milliliter polypropylene tube. Add membrane active polymer stock solution and additional NCMN Buffer A to the sample to a final concentration of 2.5%membrane active polymer and shake the solution for two hours at 20 degrees Celsius. At the end of the incubation, ultracentrifuge the sample.During the centrifugation, equilibrate a five milliliter nickel NTA column with 25 milliliters of NCMN Buffer A.At the end of the centrifugation, transfer the supernatant onto the column and use a syringe pump set to a 0.5 milliliter per minute flow rate to load the supernatant onto the column at 20 degrees Celsius. Wash the fast protein liquid chromatography machine lines with enough NCMN Buffer B to completely flush the system and connect the column to the chromatography machine. Wash the column with 30 milliliters of NCMN Buffer B at a one milliliter per minute flow rate and collect the flowthrough.Wash the column with 30 milliliters of NCMN Buffer C at a one milliliter per minute flow rate and collect the flowthrough. Elute the protein with 20 milliliters of NCMN Buffer D at a 0.5 milliliter per minute flow rate and use a fraction collector to collect one milliliter fractions of the sample, then store the protein samples at four degrees Celsius using SDS-PAGE to check the samples that correspond to the peaks observed on the fast protein liquid chromatography elution graph. To prepare negative stain grids, wrap a glass slide with filter paper with the carbon side facing up and place the grids that are going to be used for the sample preparation onto the slide.Place the glass slide with the electron microscope grids into the chamber of a glow discharger between the two electrodes and replace the glass lid making sure the lid is centered and well-sealed. Run the glow discharging machine and make sure that the purple light generated by the plasma is visible. When the machine is finished running, wait until the chamber has reached atmospheric pressure before removing the glass lid and placing the slide onto the bench.Adjust the concentration of the purified protein samples to about 0.1 milligrams per milliliter and load 3.5 microliters of the protein sample onto the 10 nanometer thick carbon grid. After one minute, use a piece of filter paper to remove the liquid from the electron microscopy grid surface and wash the grid surface three times with one three microliter droplet of water per wash. After the last water wash, wash the grid surface two more times with three microliter droplets of fresh filtered 2%uranium acetate per wash.After the last uranium acetate wash, stain the grid with a three microliter droplet of fresh filtered 2%uranium acetate for one minute. At the end of the incubation, use a new piece of filter paper to remove the droplet and air dry the grid for at least one minute before storing the grid in a grid box for later use. Here, negative stain images obtained using transmission electron microscopy as demonstrated can be observed.The negative stain image for the wild-type Acraflavin resistance channel protein B sample purified with detergent reveals a homogeneous solution of monodispersed particles with the protein displaying a well-defined trimeric quaternary structure. These trimeric structures correspond with the observed size exclusion chromatogram after purification. In comparison, in the negative stain image for the P223G mutant also purified with detergent, a heterogeneous solution of polydispersed nanoparticles with a propensity toward aggregation but no observable native trimers can be observed.These results are also supported by size exclusion chromatography. Similar results were observed for wild-type and mutant proteins after purification with the membrane active polymer NCMNP11. The use of the NCMNP52 polymer facilitates the generation of native cell membrane nanoparticles in much larger sizes allowing multiple Acraflavin resistance channel protein B trimers to be imaged in a single native cell membrane particle.In the mutant sample, however, no trimer particles are observed, even when looking at the large native cell membrane bilayer patches. SDS-PAGE analysis of the purified protein samples confirms the presence of Acraflavin resistance B in all of the samples with a greater than 95%purity at the predicted molecular weight of the protein. When modifying this protocol for other proteins, it is important to experimentally determine the appropriate amount of membrane fraction and the length and time and temperature for the solubilization process.If the images of your sample reveal a homogenous solution of monodispersed particles with well-defined structural units, the sample can be used for high-resolution structure determination with cryo-electron microscopy.

Research

JoVE Journal

Biochemistry

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

HCN1 और TRIP8b बीच प्रोटीन, प्रोटीन बातचीत के छोटे अणु inhibitors की पहचान के लिए विधि

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the ...

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

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पाड़ Liposomes का उपयोग लिपिड-समीपस्थ प्रोटीन, प्रोटीन बातचीत पुनर्गठित करने<em&gt; इन विट्रो</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

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A Modified Yeast-one Hybrid System for Heteromeric Protein Complex-DNA Interaction Studies

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Over the years, the yeast one-hybrid assay has proven to be an important technique for the identification and validation of physical interactions between ...

Biotinylated Cell-penetrating Peptides to Study Intracellular Protein-protein Interactions

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Analysis of Thylakoid Membrane Protein Complexes by Blue Native Gel Electrophoresis

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A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

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A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

Evaluation of Protein&#8211;Protein Interactions using an On-Membrane Digestion Technique

एक पर झिल्ली पाचन तकनीक का उपयोग कर प्रोटीन-प्रोटीन इंटरेक्शन का मूल्यांकन

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions ...

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

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ई. कोलाई में हेटेरोलोगस अभिव्यक्ति और झिल्ली वेसिकल्स के उत्पादन द्वारा झिल्ली ट्रांसपोर्टरों का लक्षण वर्णन

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Mitochondrial dynamics is essential for the organelle&#8217;s diverse functions and cellular responses. The crowded, spatially complex, mitochondrial ...