Name: luminophore formation in various conformations of bovine serum albumin by binding of gold(iii)
Uploaded: 2018-08-31T15:00:00.000+00:00
Duration: 8 min 26 s
Description: Watch this Scientific Journal Video about Luminophore Formation in Various Conformations of Bovine Serum Albumin by Binding of GoldIII at JoVE.com

S.E anerkennt die Unterstützung von Duke Stiftungsfonds besondere Initiative, Wells Fargo Fonds, PhRMA Stiftung sowie Startup-Fonds von der University of North Carolina, Charlotte.

(1) Synthese von BSA-Au(III) Komplex
<ol><li>25 mg der BSA in 1 mL Hochleistungs-Flüssigkeitschromatographie (HPLC) Grade Wasser in einer 5 mL Reaktion Phiole zu lösen. Hinweis: Die Lösung sollte klar angezeigt werden.</li>
	<li>Gold (III) chlorid Trihydrate (chlorogoldsäure) zu einer Konzentration von 5 mM in HPLC Grade Wasser auflösen. Hinweis: Die Lösung sollte gelb angezeigt. Chloroauric Säure Lösung vorbereitet bei dieser Konzentration führt zu einem BSA in Au Verhältnis von 01:13.<ol>
<li>Alternativ bereiten Sie eine Lösung von chlorogoldsäure mit einer Konzentration von überall zwischen 0,38 mM (BSA:Au = 1:1) bis 20 mM (BSA:Au = 01:50) in HPLC Grad Wasser. Hinweis: Unterschiedliche Verhältnisse der BSA Gold drastisch unterschiedlich rote Fluoreszenz-Muster der Erregung-Emission Karte führt.</li>
	</ol>
</li>
	<li>Die Reaktion Phiole der BSA in einem 37 ° C Wasserbad legen und kräftig rühren bei 750 u/min mit einem Magnetrührer.</li>
	<li>Unmittelbar nach der Aufregung beginnt, fügen Sie 1 mL der chlorogoldsäure der Projektmappe hinzu. Die Farbe der Lösung sollte von klar bis gelb verwandeln.</li>
	<li>Rühren Sie die Mischung für 2 min bei 37 ° C und bei 750 u/min mit einem Magnetrührer.</li>
	<li>Fügen Sie in das Fläschchen Reaktion 100 μL 1 M NaOH Lösung bringen den pH-Wert auf 12 hinzu. Hinweis: Unmittelbar nach NaOH hinzugefügt wird, die Lösung sollte leicht zu einem gelb-braun zu verdunkeln und dann zurückdrehen bis gelb.</li>
	<li>Weiterhin rühren bei 750 u/min für 2 h und bei 37 ° C. Die Lösung sollte langsam von Gelb in eine dunkle gelb/braun Farbe ändern. Diese Farbveränderung zeigt die Bildung der roten fluoreszierenden BSA-Au(III) komplexe.</li>
	<li>Lassen Sie die Probe bei Raumtemperatur für 2 Tage und die Lösung zu sitzen wird weiterhin ein Bernstein Braun verdunkeln und die Fluoreszenzintensität erhöht.<ol>
<li>Alternativ lassen Sie die Probe bei 37 ° C für 12 rühren mehr h wie die Farbe der Lösung zu einem braunen Bernstein entwickelt sich weiter.</li>
	</ol>
</li>
</ol>(2) Synthese von BSA-Cu(II)-Au(III)
<ol><li>25 mg der BSA in 1 mL HPLC Grade Wasser auflösen. Die Lösung sollte klar erscheinen.</li>
	<li>Kupfer (II)-chlorid-Dihydrat in HPLC Grade Wasser zu einer Konzentration von 5 mM zu lösen. Die Lösung sollte Licht erscheinen blau.</li>
	<li>Eine Reaktion Ampulle 5 mL 1 mL der wässrigen BSA-Lösung hinzu und legen Sie in einem Wasserbad bei 37 ° C. Rühren Sie die Mischung bei 750 u/min.</li>
	<li>Sofort die Reaktion Phiole 0,5 mL Kupfer (II)-chlorid-Dihydrat-Lösung hinzu und mischen Sie 2 min. lang. Die Lösung bleibt Licht blau.</li>
	<li>Fügen Sie 75 μL 1 M NaOH bis pH 12 und 2 h zu mischen. Die Lösung wird lila werden.</li>
	<li>Lösen Sie chlorogoldsäure in HPLC Grade Wasser zu einer Konzentration von 5 mM auf.</li>
	<li>Die Reaktion Phiole 0,5 mL wässrige chlorogoldsäure hinzu und passen Sie den pH-Wert wieder auf 12 mit 1 M NaOH.</li>
	<li>Rühren Sie das Reaktionsgemisch für 2 h. Hinweis: Die Lösung sollte auf eine braune Farbe zu entwickeln.</li>
</ol>(3) Synthese von BSA-Ni(II)-Au(III)
<ol><li>25 mg der BSA in 1 mL HPLC Grade Wasser auflösen. Die Lösung sollte klar erscheinen.</li>
	<li>Nickel (II)-Chlorid-Hexahydrat in HPLC Grade Wasser zu einer Konzentration von 5 mM zu lösen. Die Lösung sollte Licht erscheinen grün.</li>
	<li>Eine Reaktion Ampulle 5 mL 1 mL der wässrigen BSA-Lösung hinzu und legen Sie in einem Wasserbad bei 37 oC. rühren die Mischung bei 750 u/min.</li>
	<li>Fügen Sie 0,5 mL der Nickel (II)-Chlorid-Hexahydrat-Lösung sofort die Reaktion Phiole und Mischung für 2 min hinzu. Hinweis: Die Lösung bleibt Licht grün.</li>
	<li>Fügen Sie 75 μL 1 M NaOH bis pH 12 und 2 h zu mischen. Hinweis: Die Lösung werden dunkelgelb.</li>
	<li>Lösen Sie chlorogoldsäure in HPLC Grade Wasser zu einer Konzentration von 5 mM auf.</li>
	<li>Die Reaktion Phiole 0,5 mL der wässrigen chlorogoldsäure hinzu und passen Sie den pH-Wert wieder auf 12.</li>
	<li>Rühren Sie das Reaktionsgemisch für 2 h. Hinweis: Die Lösung sollte auf eine braune Farbe zu entwickeln.</li>
</ol>4. Synthese von [Cys34-capped-BSA]-Au(III)
<ol><li>2 mg N-Ethylmaleimide (NEM) in 1 mL Phosphat gepufferte Kochsalzlösung (PBS, pH 7,4) auflösen.</li>
	<li>2 mg der BSA in 1 mL PBS-NEM Lösung zu lösen.</li>
	<li>Die Lösung zu einem 5 mL Reaktion Fläschchen zu übertragen und bei 20 ° C bei 500 u/min für 1 h rühren.</li>
	<li>Dialyse der Lösung mit 12 kDa Dialyse Schläuche in 500 mL PBS, bei 50 u/min unter Rühren mit einem Magnetrührer über Nacht nicht umgesetztes NEM entfernen.</li>
	<li>Lösen Sie chlorogoldsäure mit PBS-Puffer zu einer Konzentration von 0,4 mM auf. Hinweis: Die Lösung wird ein schwaches gelb sein.</li>
	<li>Übertragung der Reaktion Fläschchen in einem Wasserbad bei 37 ° C. Bei 750 u/min rühren.</li>
	<li>Sofort die Reaktion Ampulle 1 mL Chloroauric Säure-Lösung hinzufügen und für 2 min mischen lassen.</li>
	<li>Das Fläschchen Reaktion den pH 12 und für 2 h mischen fügen Sie 75 μL 1 M NaOH hinzu.</li>
</ol>5. Synthese von [all-thiol-capped-BSA]-Au(III)
<ol><li>Bereiten Sie eine Lösung von 2 M Harnstoff und 50 mM Ammonium Bicarbonat (NH4HCO3, pH 8,0) in HPLC Grade Wasser.</li>
	<li>3,3 mg der BSA in 1 mL der obigen Lösung und Transfer zu einem 5 mL-Fläschchen Reaktion auflösen.</li>
	<li>Machen Sie eine Stammlösung von 0,25 M tris(2-carboxyethyl) Phosphin (DÄMMUND) durch DÄMMUND 62,5 mg in 1 mL HPLC Wasser auflösen.</li>
	<li>Die Reaktion Phiole fügen Sie der Stammlösung DÄMMUND bis die Endkonzentration von DÄMMUND 8 mM ist hinzu.</li>
	<li>Inkubieren Sie die Lösung in einem Wasserbad für 1 h bei 50 ° c Rühren Sie bei 500 u/min mit einem Magnetrührer.</li>
	<li>Lassen Sie die Lösung vollständig auf Zimmertemperatur abkühlen.</li>
	<li>Bereiten Sie eine Stammlösung von 100 mM NEM durch NEM 12,5 mg in 1 mL HPLC Grade Wasser auflösen.</li>
	<li>Die Reaktion Phiole fügen Sie der Stammlösung von NEM bis die Endkonzentration von NEM 16 mM ist hinzu.</li>
	<li>Lassen Sie die Lösung verbinden für 2 h bei 20 ° C. Bei 500 u/min rühren.</li>
	<li>Dialyse die Lösung unter Verwendung eines 12 kDa Dialyse Schlauchs, rühren die Lösung bei 50 u/min mit einem Magnetrührer über Nacht in 500 mL 50 mM NH4HCO3 , überschüssige DÄMMUND, NEM und Harnstoff zu entfernen.</li>
	<li>Bewegen Sie die Reaktion Phiole in einem Wasserbad bei 37 ° C und bei 750 u/min rühren.</li>
	<li>Chlorogoldsäure in HPLC Grade Wasser zu einer Konzentration von 0,66 mM auflösen.</li>
	<li>Sofort die Reaktion Ampulle 1 mL Chloroauric Säure-Lösung hinzufügen und für 2 min mischen lassen.</li>
	<li>Fügen Sie 1 M NaOH bis pH der Lösung 12 und lassen Sie die Lösung weiterhin für 2 h mischen.</li>
</ol>

<ol>
	<li>Xie, J., Zheng, Y., Ying, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-Directed+Synthesis+of+Highly+Fluorescent+Gold+Nanoparticles.">Protein-Directed Synthesis of Highly Fluorescent Gold Nanoparticles.</a> Journal of the American Chemical Society. 131, 888-889 (2009).</li><li>Saha, K., Agasti, S. S., Kim, C., Li, X., Rotello, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gold+Nanoparticles+in+Chemical+and+Biological+Sensing.">Gold Nanoparticles in Chemical and Biological Sensing.</a> Chemical Reviews. 112, 2739-2779 (2012).</li><li>Zhang, Y., 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=New+Gold+Nanostructures+for+Sensor+Applications:+A+Review.">New Gold Nanostructures for Sensor Applications: A Review.</a> Materials. 7, 5169-5201 (2014).</li><li>Chen, L. -. Y., Wang, C. -. W., Yuan, Z., Chang, H. -. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+Gold+Nanoclusters:+Recent+Advances+in+Sensing+and+Imaging.">Fluorescent Gold Nanoclusters: Recent Advances in Sensing and Imaging.</a> Analytical Chemistry. 87 (1), 216-229 (2015).</li><li>Cai, W., Gao, T., Hong, H., Sun, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+Gold+Nanoparticles+in+Cancer+Nanotechnology.">Applications of Gold Nanoparticles in Cancer Nanotechnology.</a> Nanotechnology, Science and Applications. 1, 17-32 (2008).</li><li>Nune, S. 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=Nanoparticles+for+Biomedical+Imaging.">Nanoparticles for Biomedical Imaging.</a> Expert Opinion on Drug Delivery. 6, 1175-1194 (2009).</li><li>Dorsey, J. 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=Gold+Nanoparticles+in+Radiation+Research:+Potential+Applications+for+Imaging+and+Radiosensitization.">Gold Nanoparticles in Radiation Research: Potential Applications for Imaging and Radiosensitization.</a> Translational Cancer Research. 2, 280-291 (2013).</li><li>Daniel, M. -. C., Astruc, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gold+Nanoparticles:+Assembly,+Supramolecular+Chemistry,+Quantum-Size-Related+Properties,+and+Applications+toward+Biology,+Catalysis,+and+Nanotechnology.">Gold Nanoparticles: Assembly, Supramolecular Chemistry, Quantum-Size-Related Properties, and Applications toward Biology, Catalysis, and Nanotechnology.</a> Chemical Reviews. 104 (1), 293-346 (2004).</li><li>Ferrari, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+Nanotechnology:+Opportunities+and+Challenges.">Cancer Nanotechnology: Opportunities and Challenges.</a> Nature Reviews Cancer. 5, 161-171 (2005).</li><li>Huang, X., Jain, P. K., El-Sayed, I. H., El-Sayed, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gold+Nanoparticles:+Interesting+Optical+Properties+and+Recent+Applications+in+Cancer+Diagnostics+and+Therapy.">Gold Nanoparticles: Interesting Optical Properties and Recent Applications in Cancer Diagnostics and Therapy.</a> Nanomedicine. 2, 681 (2007).</li><li>Arvizo, R., Bhattacharya, R., Mukherjee, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gold+Nanoparticles:+Opportunities+and+Challenges+in+Nanomedicine.">Gold Nanoparticles: Opportunities and Challenges in Nanomedicine.</a> Expert Opinion on Drug Delivery. 7, 753-763 (2010).</li><li>Doane, T. L., Burda, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Unique+Role+of+Nanoparticles+in+Nanomedicine:+Imaging,+Drug+Delivery+and+Therapy.">The Unique Role of Nanoparticles in Nanomedicine: Imaging, Drug Delivery and Therapy.</a> Chemical Society Reviews. 41, 2885 (2012).</li><li>Egusa, S., Ebrahem, Q., Mahfouz, R. Z., Saunthararajah, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ligand+Exchange+on+Gold+Nanoparticles+for+Drug+Delivery+and+Enhanced+Therapeutic+Index+Evaluated+in+Acute+Myeloid+Leukemia+Models.">Ligand Exchange on Gold Nanoparticles for Drug Delivery and Enhanced Therapeutic Index Evaluated in Acute Myeloid Leukemia Models.</a> Experimental Biology and Medicine. 239, 853 (2014).</li><li>Qu, X., 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=Fluorescent+Gold+Nanoclusters:+Synthesis+and+Recent+Biological+Application.">Fluorescent Gold Nanoclusters: Synthesis and Recent Biological Application.</a> Journal of Nanomaterials. (784097), (2015).</li><li>Chakraborty, I., Pradeep, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomically+Precise+Clusters+of+Noble+Metals:+Emerging+Link+between+Atoms+and+Nanoparticles.">Atomically Precise Clusters of Noble Metals: Emerging Link between Atoms and Nanoparticles.</a> Chemical Reviews. 117, 8208-8271 (2017).</li><li>Raut, 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=Evidence+of+energy+transfer+from+tryptophan+to+BSA/HSA+protected+gold+nanoclusters.">Evidence of energy transfer from tryptophan to BSA/HSA protected gold nanoclusters.</a> Methods and Applications in Fluorescence. 2, (2014).</li><li>Le Gu&#233;vel, X., Daum, N., Schneider, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+Characterization+of+Human+Transferrin-Stabilized+Gold+Nanoclusters.">Synthesis and Characterization of Human Transferrin-Stabilized Gold Nanoclusters.</a> Nanotechnology. 22 (27), (2011).</li><li>Kawasaki, H., Yoshimura, K., Hamaguchi, K., Arakawa, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypsin-Stabilized+Fluorescent+Gold+Nanocluster+for+Sensitive+and+Selective+Hg2++Detection.">Trypsin-Stabilized Fluorescent Gold Nanocluster for Sensitive and Selective Hg2+ Detection.</a> Analytical Sciences. 27 (6), 591 (2011).</li><li>Lu, D., 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=Lysozyme-Stabilized+Gold+Nanoclusters+as+a+Novel+Fluorescence+Probe+for+Cyanide+Recognition.">Lysozyme-Stabilized Gold Nanoclusters as a Novel Fluorescence Probe for Cyanide Recognition.</a> Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy. 121, 77-80 (2014).</li><li>Dixon, J. M., Egusa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+Change-Induced+Fluorescence+of+Bovine+Serum+Albumin-Gold+Complexes.">Conformational Change-Induced Fluorescence of Bovine Serum Albumin-Gold Complexes.</a> Journal of the American Chemical Society. 140, 2265-2271 (2018).</li><li>Peters, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> All About Albumin. , (1996).</li><li>Masuoka, J., Saltman, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zinc(II)+and+Copper(II)+Binding+to+Serum+Albumin.+A+Comparative+Study+of+Dog,+Bovine,+and+Human+Albumin.">Zinc(II) and Copper(II) Binding to Serum Albumin. A Comparative Study of Dog, Bovine, and Human Albumin.</a> Journal of Biological Chemistry. 269, 25557-25561 (1994).</li><li>Takeda, K., Wada, A., Yamamoto, K., Moriyama, Y., Aoki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+Change+of+Bovine+Serum+Albumin+by+Heat+Treatment.">Conformational Change of Bovine Serum Albumin by Heat Treatment.</a> Journal of Protein Chemistry. 8 (5), 653-659 (1989).</li><li>Klotz, I. M., Curme, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Thermodynamics+of+Metallo-protein+Combinations.+Copper+with+Bovine+Serum+Albumin.">The Thermodynamics of Metallo-protein Combinations. Copper with Bovine Serum Albumin.</a> Journal of the American Chemical Society. 70, 939-943 (1948).</li><li>Fiess, H. A., Klotz, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Thermodynamics+of+Metallo-Protein+Combinations.+Comparison+of+Copper+Complexes+with+Natural+Proteins.">The Thermodynamics of Metallo-Protein Combinations. Comparison of Copper Complexes with Natural Proteins.</a> J. Am. Chem. Soc. 74, 887-891 (1952).</li><li>Rao, M. S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Study+of+the+Interaction+of+Nickel(II)+with+Bovine+Serum+Albumin.">A Study of the Interaction of Nickel(II) with Bovine Serum Albumin.</a> Journal of the American Chemical Society. 84, 1788-1790 (1962).</li><li>Peters, T., Blumenstock, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Copper-Binding+Properties+of+Bovine+Serum+Albumin+and+Its+Amino-terminal+Peptide+Fragment.">Copper-Binding Properties of Bovine Serum Albumin and Its Amino-terminal Peptide Fragment.</a> Journal of Biological Chemistry. 242, 1574-1578 (1967).</li><li>Xue, Y., Li, X., Li, H., Zhang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+Thiol-Gold+Interactions+towards+the+Efficient+Strength+Control.">Quantifying Thiol-Gold Interactions towards the Efficient Strength Control.</a> Nature Communications. 5, 4348 (2014).</li><li>Xu, Y., 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+Role+of+Protein+Characteristics+in+the+Formation+and+Fluorescence+of+Au+Nanoclusters.">The Role of Protein Characteristics in the Formation and Fluorescence of Au Nanoclusters.</a> Nanoscale. 6 (3), 1515-1524 (2014).</li></ol>

Die Autoren haben nichts preisgeben.

Die BSA-Au(III) Verbindungen vorbereitet bei pH 12 weisen rote Fluoreszenz bei einer Emissionswellenlänge λEm= 640 nm bei Erregung mit ultravioletten (UV) Licht λex= 365 nm (Abbildung 1A). Die Entstehung der rote Fluoreszenz ist ein langsamer Prozess und dauert ein paar Tage bei Raumtemperatur bis zu einer maximalen Intensität zu erhöhen. Läuft die Reaktion bei 37 ° C wird die optimale Ergebnisse liefern, obwohl höherer Temperatur verwendet werden kann,...

Eine BSA-Au-Verbindung Ausstellen einer Ultraviolett (UV)-leicht erregbaren rote Fluoreszenz, mit bemerkenswerten schürt Verschiebung, wurde ursprünglich von Xie Et al.synthetisiert worden. 1. einzigartige und stabile rote Fluoreszenz finden Sie verschiedene Anwendungen in den Bereichen Fernerkundung,2,3,4, bildgebenden5,6,7oder Nanomedizin8 ,9,10,11,12,13. Diese Verbindung wurde von vielen Forschern auf dem Gebiet der Nano-Wissenschaft in den letzten Jahren14,15,16ausgiebig untersucht. Die BSA-Au-Verbindung wurde als Au25 nanocluster interpretiert. Das Ziel der vorgestellten Methode ist, diese Verbindung im Detail zu untersuchen und zu verstehen, die Herkunft der rote Fluoreszenz. Den vorgestellten Ansatz folgend können das Vorhandensein von mehreren Au Bindungsstellen, und die Herkunft der Fluoreszenz, Alternative zu einem Standort Nukleation von Au25 nanocluster dargestellt werden. Der gleiche Ansatz kann eingesetzt werden, zu studieren, wie andere Proteine17,18,19 komplexiert mit Au(III) ihren fluoreszierenden Eigenschaften ändern können.
Die Synthese der BSA-Au rot fluoreszierende Verbindung erfordert eine enge Kontrolle über das molare Verhältnis von BSA, Au (BSA:Au), die Intensität der Fluoreszenz und die Lage der Gipfel der Erregung-Emission Karte (EEM)20zu maximieren. Es kann gezeigt werden, dass mehrere Bindungsstellen für Au(III) zu binden, gibt es einschließlich der Asparagin-Fragment (oder Asp-Fragment, die ersten vier Aminosäurereste am N-Terminus des BSA)21,22. Die 34th Aminosäure der BSA (Cys-34) zeigt sich auch, Au(III) zu koordinieren und in den Mechanismus der roten fluorescence([Cys34-capped-BSA]-Au(III))20einbezogen werden. Auf alle Cys-Cys-Disulfid-Bindungen zu Spalten und verschließen alle Thiole, rote Fluoreszenz ist nicht produziert ([all-thiol-capped-BSA]-Au(III)). Dies weist auf die Notwendigkeit der Cys-Cys Disulfid Anleihen als die Au(III)-Bindungsstelle, die rote Fluoreszenz zu produzieren.
Protein-Chemie-Techniken haben nicht weit verbreitet, um die BSA-Au(III)-komplexe in der Nano-Science-Community zu studieren. Allerdings wäre es wertvoll, um diese Techniken zu verstehen, bestimmte Aspekte dieser komplexe, Detailwissen über die Au(III)-Bindungsstellen im BSA zu gewinnen. Dieser Artikel soll einige dieser Techniken zu zeigen.

<table><tbody><tr><td>Bovine Serum Albumin (BSA), 96%</td><td>Sigma-Aldrich</td><td>A5611</td><td></td></tr><tr><td>gold (III) chloride trihydrate, 99.9%</td><td>Sigma-Aldrich</td><td>520918</td><td></td></tr><tr><td>Copper (II) chloride dihydrate, 99.999%</td><td>Sigma-Aldrich</td><td>459097</td><td></td></tr><tr><td>Nickel (II) chloride hexahydrate, 99.9%</td><td>Sigma-Aldrich</td><td>654507</td><td></td></tr><tr><td>N-Ethylmaleimide (NEM), &gt;99.0%</td><td>Sigma-Aldrich</td><td>4259</td><td></td></tr><tr><td>Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), &gt;98.0%</td><td>Sigma-Aldrich</td><td>C4706</td><td></td></tr><tr><td>Sodium hydroxide, &gt;98.0%</td><td>Sigma-Aldrich</td><td>S8045</td><td></td></tr><tr><td>Urea, 99.5%</td><td>Chem-Implex Int&#039;l</td><td>30142</td><td></td></tr><tr><td>Phospate buffered saline (PBS)</td><td>Corning</td><td>MT21040CV</td><td></td></tr><tr><td>Ammonium bicarbonate, 99.5%</td><td>Sigma-Aldrich</td><td>9830</td><td></td></tr></tbody></table>

luminophore formation in various conformations of bovine serum albumin by binding of gold(iii)

Die vorgestellten Protokolle soll den Prozess der Au(III) Bindung an die BSA, nachgiebig Konformation bedingte rote Fluoreszenz (λEm = 640 nm) der BSA-Au(III)-komplexe. Die Methode stellt den pH-Wert zu zeigen, dass die Entstehung der rote Fluoreszenz mit der pH-induzierte Gleichgewicht Übergänge von der BSA Konformationen korreliert ist. Rot fluoreszierende BSA-Au(III)-komplexe können nur mit einer Einstellung des pH-Wertes bei oder über 9,7, das entspricht der "A-Form" Konformation der BSA gebildet werden. Das Protokoll die BSA, Au Molverhältnis anpassen und den zeitlichen Verlauf des Prozesses der Au(III) Bindung zu überwachen ist beschrieben. Die minimale Anzahl von Au(III) pro BSA, der rote Fluoreszenz zu produzieren ist weniger als sieben. Wir beschreiben das Protokoll in Schritten, um das Vorhandensein von mehreren Au(III)-Bindungsstellen im BSA zu veranschaulichen. Erstens, durch Zugabe von Kupfer (Cu(II)) oder Nickel (Ni(II)) kationen gefolgt von Au(III), zeigt diese Methode eine Bindungsstelle für Au(III), die nicht das rote Fluorophor. Zweitens wird durch BSA von Thiol Deckelung Agenten ändern, ein anderes Nonfluorophore bilden Au(III) Bindungsstelle aufgedeckt. Drittens sind ändern die BSA-Konformation durch Spaltung und Begrenzung der Disulfid-Bindungen, die mögliche Au(III) Bindung Webseite(n) illustriert. Das Protokoll beschrieben, zur Steuerung der BSA Konformationen und Au(III) Bindung, kann in der Regel angewendet werden, um die Wechselwirkungen von anderen Proteinen und Metallkationen zu studieren.

Aus der Fluoreszenz des BSA-Au(III) komplexes, es wurde beobachtet, dass die Umwandlung der inneren blauen Fluoreszenz der BSA (λEm = 400 nm), rote Fluoreszenz (λEm = 640 nm) tritt bei etwa pH 9,7 durch ein Gleichgewicht Übergang (Abbildung 1). EEM BSA-Au(III) bei verschiedenen BSA Au molare Verhältnis ist in Abbildung 2dargestellt, und diese Daten zeigt, wie die gleichen Emissionswelle...

Watch this Scientific Journal Video about Luminophore Formation in Various Conformations of Bovine Serum Albumin by Binding of GoldIII at JoVE.com

Luminophore Formation in Various Conformations of Bovine Serum Albumin by Binding of GoldIII

Die Protokolle für das Studium der Bindung von gold-Kationen (Au(III)) zu verschiedenen Konformationen der Rinderserumalbumin (BSA) wie auch für die Charakterisierung der Konformationsänderungen abhängigen einzigartige BSA-Au Fluoreszenz dargestellt werden.

Luminophor Bildung in verschiedenen Konformationen von Rinderserumalbumin durch Bindung von Gold(III)

The purpose of the presented protocols is to study the process of Au(III) binding to BSA, yielding conformation change-induced red fluorescence (&#955;em ...

luminophore-formation-various-conformations-bovine-serum-albumin

Research

JoVE Journal

Chemistry

Luminophore Formation in Various Conformations of Bovine Serum Albumin by Binding of Gold(III)

6.8K Aufrufe.  University of North Carolina. Die Protokolle für das Studium der Bindung von gold-Kationen (Au(III)) zu verschiedenen Konformationen der Rinderserumalbumin (BSA) wie auch für die Charakterisierung der Konformationsänderungen abhängigen einzigartige BSA-Au Fluoreszenz dargestellt werden.

Serie: Luminophore Formation in Various Conformations of Bovine Serum Albumin by Binding of GoldIII

Iridium(III) Luminescent Probe for Detection of the Malarial Protein Biomarker Histidine Rich Protein-II

This work outlines the synthesis of a non-emissive, cyclometalated Ir(III) complex, Ir(ppy)2(H2O)2+ (Ir1), which elicits a rapid, long-lived phosphorescent signal when coordinated to a histidine-containing protein immobilized on the surface of a magnetic particle. Synthesis of Ir1, in high yields,is complete O/N&#160;and involves splitting of the parent cyclometalated Ir(III) chloro-bridged dimer into two equivalents of the solvated complex. To confirm specificity, several amino acids were probed for coordination activity when added to the synthesized probe, and only histidine elicited a signal response. Using BNT-II, a branched peptide mimic of the malarial biomarker Histidine Rich Protein II (pfHRP-II), the iridium probe was validated as a tool for HRP-II detection. Quenching effects were noted in the BNT-II/Ir1 titration when compared to L-Histidine/Ir1, but these were attributed to steric hindrance and triplet state quenching. Biolayer interferometry was used to determine real-time kinetics of interaction of Ir1 with BNT-II. Once the system was optimized, the limit of detection of rcHRP-II using the probe was found to be 12.8 nM in solution. When this protein was immobilized on the surface of a 50 &#181;m magnetic agarose particle, the limit of detection was 14.5 nM. The robust signal response of this inorganic probe, as well as its flexibility of use in solution or immobilized on a surface, can lend itself toward a variety of applications, from diagnostic use to imaging.

IridiumIII Luminescent Probe for Detection of the Malarial Protein Biomarker Histidine Rich Protein-II

Robust detection reagents are of increasing necessity for developing new malaria diagnostic tools. An iridium(III) probe was designed that emits long-lasting luminescent signal in the presence of a histidine-rich malarial protein biomarker. Detection of the protein either in solution or immobilized on a magnetic particle affords flexibility in application.

Iridium (III) Lumineszierende Sonde zum Nachweis von Malaria-Protein Biomarker Histidin Rich Protein-II

This work outlines the synthesis of a non-emissive, cyclometalated Ir(III) complex, Ir(ppy)2(H2O)2+ (Ir1), which elicits a rapid, long-lived ...

Forschung

Chemie

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a fluorescent dye and are spotted by a photodetector following their separation. Single molecule fluorescence imaging can provide ultrasensitive protein detection with its ability for visualizing individual fluorescent molecules. However, the application of this powerful imaging method to electrophoresis assays is hampered by the lack of ways to characterize the homogeneity of fluorescent labeling of each protein species across the proteome. Here, we developed a method to evaluate the labeling homogeneity across the proteome based on a single molecule fluorescence imaging assay. In our measurement using a HeLa cell sample, the proportion of proteins labeled with at least one dye, which we termed &#8216;labeling occupancy&#8217; (LO), was determined to range from 50% to 90%, supporting the high potential of the application of single molecule imaging to sensitive and precise proteome analysis.

Here, we present a protocol to assess the labeling homogeneity for each protein species in a complex protein sample at the single molecule level.

Proteom-weite Quantifizierung des Benennens Homogenität auf der Ebene des einzelnen Moleküls

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

Biochemie

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM)

Protein-protein interactions (PPIs) are key molecular events to biology. However, it remains a challenge to visualize PPIs with sufficient resolution and sensitivity in cells because the resolution of conventional light microscopy is diffraction-limited to ~250 nm. By combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM), PPIs can be visualized in cells with single molecule sensitivity and nanometer spatial resolution. BiFC is a commonly used technique for visualizing PPIs with fluorescence contrast, which involves splitting of a fluorescent protein into two non-fluorescent fragments. PALM is a recent superresolution microscopy technique for imaging biological samples at the nanometer and single molecule scales, which uses phototransformable fluorescent probes such as photoactivatable fluorescent proteins (PA-FPs). BiFC-PALM was demonstrated by splitting PAmCherry1, a PA-FP compatible with PALM, for its monomeric nature, good single molecule brightness, high contrast ratio, and utility for stoichiometry measurements. When split between amino acids 159 and 160, PAmCherry1 can be made into a BiFC probe that reconstitutes efficiently at 37 &#176;C with high specificity to PPIs and low non-specific reconstitution. Ras-Raf interaction is used as an example to show how BiFC-PALM helps to probe interactions at the nanometer scale and with single molecule resolution. Their diffusion can also be tracked in live cells using single molecule tracking (smt-) PALM. In this protocol, factors to consider when designing the fusion proteins for BiFC-PALM are discussed, sample preparation, image acquisition, and data analysis steps are explained, and a few exemplary results are showcased. Providing high spatial resolution, specificity, and sensitivity, BiFC-PALM is a useful tool for studying PPIs in intact biological samples.

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation BiFC-PALM

Protein-protein interactions are visualized in cells with nanometer spatial resolution by combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM). Described here is the use of BiFC-PALM for imaging Ras-Raf interactions in U2OS cells for visualizing the nanoscale clustering and diffusion of individual Ras-Raf complexes.

Photoaktivierte Lokalisationsmikroskopie mit Bimolekulare Fluoreszenzkomplementation (BIFC-PALM)

Protein-protein interactions (PPIs) are key molecular events to biology. However, it remains a challenge to visualize PPIs with sufficient resolution and ...

Biotechnik

A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold nanoclusters (Au NCs) within the drug-loaded protein, based on the differential fluorescence signal emitted by the Au NCs. Albumin proteins such as human serum albumin (HSA) and bovine serum albumin (BSA) are selected as the model proteins. Four small molecular drugs (e.g., ibuprofen, warfarin, phenytoin, and sulfanilamide) of different binding affinities to the albumin proteins are tested. It was found that the formation rate of fluorescent Au NCs inside the drug loaded albumin protein under denaturing conditions (i.e., 60 &#176;C or in the presence of urea) is slower than that formed in the pristine protein (without drugs). Moreover, the fluorescent intensity of the as-formed NCs is found to be inversely correlated to the binding affinities of these drugs to the albumin proteins. Particularly, the higher the drug-protein binding affinity, the slower the rate of Au NCs formation, and thus a lower fluorescence intensity of the resultant Au NCs is observed. The fluorescence intensity of the resultant Au NCs therefore provides a simple measure of the relative binding strength of different drugs tested. This method is also extendable to measure the specific drug-protein binding constant (KD) by simply varying the drug content preloaded in the protein at a fixed protein concentration. The measured results match well with the values obtained using other prestige but more complicated methods.

A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

Eine schnelle und quantitative fluorimetrische Verfahren zur Protein-Targeting kleines Molekül als Wirkstoff-Screening

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold ...

Determining Total Protein and Bioactive Protein Concentrations in Bovine Colostrum

Colostrum is a complex biological fluid produced by mammals immediately after parturition. It meets all the nutritional requirements for neonates as a good source of macro- and micronutrients, bioactive peptides, and growth factors. Bovine colostrum is also a potential source of nutrition and bioactive because of its rich protein content that includes immunoglobulin G (IgG) and lactoferrin. However, the level of lactoferrin and IgG in bovine colostrum changes markedly during the lactation period. Therefore, monitoring the concentration of IgG and lactoferrin for the use of bovine colostrum as a protein source is an important question to study. Methods in this article describe how to determine protein content, as well as specific concentrations of lactoferrin and IgG. These methods include the following steps: Isolation of bovine colostrum proteins, Determination of protein concentration via Bicinchoninic acid assay (BCA), Visualization of proteins via SDS-PAGE, Determination of lactoferrin, and IgG concentration using an ELISA Assay.

Bovine colostrum is both a primary source of nutrients and immunological support for the newborn calf. The understanding of the level of therapeutic proteins (lactoferrin and IgG) is important for the bovine colostrum dosing and standardization for human consumption.

Colostrum is a complex biological fluid produced by mammals immediately after parturition. It meets all the nutritional requirements for neonates as a ...

Limited Proteolysis and Gel Electrophoresis in the Presence of Metal Cations: Au(III)-binding Luminescent Domain in Serum Albumins

The purpose of the presented protocols is to determine the domain of Au(III) binding in BSA. The BSA-Au(III) compound exhibits ultraviolet (UV)-excitable red luminescence (&#955;em = 640 nm), with unusual Stokes shifts compared to the innate UV/blue fluorescence arising from the aromatic residues. Red-luminescent complexes are formed in highly alkaline conditions above pH 10 and require a conformation change within the protein to occur. In addition, preservation of Cys-Cys disulfide bonds in BSA is necessary to obtain this red luminescence. In order to understand the mechanism of this luminescence, elucidation of the luminophore-forming Au(III) binding site is essential. A facile way to assess the luminophore-forming site would be to (1) predictably fragment the protein by enzymatic digestion, (2) react the obtained fragments with Au(III), then (3) perform gel electrophoresis to observe the well-separated fragment bands and analyze the in-gel red luminescence. However, due to the alkaline conditions and the reaction with metal cations, new limited proteolysis techniques and gel electrophoresis conditions must be applied. Particularly, the presence of metal cations in gel electrophoresis can make the band separations technically difficult. We describe this new protocol in steps to identify the red-luminophore-forming metal binding domain in BSA. This protocol can thus be applied for analyzing protein fragments that must remain in a non-denatured or a partially denatured state, in the presence of metal cations. Because the majority of proteins need metal cations to function, analyses of metal-bound proteins are often desired, which have relied on x-ray crystallography in the literature. This method, on the other hand, could be used in supplement to study the interactions of proteins with metal cations without requiring the protein crystallization and at a desired pH condition.

Limited Proteolysis and Gel Electrophoresis in the Presence of Metal Cations: AuIII-binding Luminescent Domain in Serum Albumins

We present a protocol for studying the binding domain of Au(III) in bovine serum albumin (BSA).

The purpose of the presented protocols is to determine the domain of Au(III) binding in BSA. The BSA-Au(III) compound exhibits ultraviolet (UV)-excitable ...

Synthesis of Near-Infrared Emitting Gold Nanoclusters for Biological Applications

Over the past decade, fluorescent gold nanoclusters (AuNCs) have witnessed growing popularity in biological applications and enormous efforts have been devoted to their development. In this protocol, a recently developed, facile method for preparation of water soluble, biocompatible, and colloidally stable near-infrared emitting AuNCs have been described in detail. This room-temperature, bottom-up chemical synthesis provides easily functionalizable AuNCs capped with thioctic acid and thiol-modified polyethylene glycol in aqueous solution. The synthetic approach requires neither organic solvents or additional ligand exchange nor extensive knowledge of synthetic chemistry to reproduce. The resulting AuNCs offer free surface carboxylic acids, which can be functionalized with various biological molecules bearing a free amine group without adversely affecting the photoluminescent properties of the AuNCs. A quick, reliable procedure for flow cytometric quantification and confocal microscopic imaging of AuNC uptake by HeLa cells also been described. Due to the large Stokes shift, proper setting of filters in flow cytometry and confocal microscopy is necessary for efficient detection of near-infrared photoluminescence of AuNCs.

A reliable and easily reproducible method for preparation of functionalizable, near-infrared emitting photoluminescent gold nanoclusters and their direct detection inside HeLa cells by flow cytometry and confocal laser scanning microscopy is described.

Synthese von Nahinfrarot emittierenden Gold-Nanoclustern für biologische Anwendungen

Over the past decade, fluorescent gold nanoclusters (AuNCs) have witnessed growing popularity in biological applications and enormous efforts have been ...

Characterization of pH-Dependent Reversible Self-Assembly of Amyloid Beta 1-40-Coated Gold Colloids

The characterization of A&#946;1-40 coated over the nano-gold colloidal particle surfaces was conducted using surface plasmon resonance (SPR) spectroscopy and transmission electron microscopy (TEM). The observed pH-dependent shift of the SPR band of A&#946;1-40-coated 20 nm gold particles was correlated with alternation of aggregation and disaggregation observed in the TEM images. A pH of ~4&#160;induced an unfolded conformation, and a pH of ~10&#160;induced a folded conformation of A&#946;1-40 on the gold surface. This reversible aggregation process was observed by Raman imaging as the pH was gradually changed from pH 4 to pH 10. We observed a pH-dependent morphology change in A&#946;1-40 on the gold surface, where clear aggregates were observed at pH 4. However, we observed very subtle differences in the surface-enhanced Raman spectroscopy (SERS) spectrum between pH 4 and pH 10 conditions, with the most striking difference being spectral density in the region of 250 cm-1 and 1750 cm-1. Specifically, the mode analysis of the reversible aggregation indicated that the aggregates were formed by the unfolded conformation of A&#946;1-40 which involved the benzene ring section of Tyrosine and Phenylalanine. Conversely, the disassembly of the aggregates was associated with conformational changes in protein folding of A&#946;1-40 involving Histidine, Glutamine, Methionine, and Aspartic acid.

Here, we present a systematic method describing the quasi-reversible self-assembly of the amyloid beta 1-40 (A&#946;1-40)-coated 20 nm gold aggregates. The nano-size-dependent quasi-reversible networking between peptides was correlated with specific amino acids or sections of the A&#946;1-40 monomer.

Charakterisierung der pH-abhängigen reversiblen Selbstorganisation von Amyloid-beta-1-40-beschichteten Goldkolloiden

The characterization of A&#946;1-40 coated over the nano-gold colloidal particle surfaces was conducted using surface plasmon resonance (SPR) spectroscopy ...

Real-time Monitoring of Ligand-receptor Interactions with Fluorescence Resonance Energy Transfer

FRET is a process whereby energy is non-radiatively transferred from an excited donor molecule to a ground-state acceptor molecule through long-range dipole-dipole interactions1. In the present sensing assay, we utilize an interesting property of PDA: blue-shift in the UV-Vis electronic absorption spectrum of PDA (Figure 1) after an analyte interacts with receptors attached to PDA2,3,4,7. This shift in the PDA absorption spectrum provides changes in the spectral overlap (J) between PDA (acceptor) and rhodamine (donor) that leads to changes in the FRET efficiency. Thus, the interactions between analyte (ligand) and receptors are detected through FRET between donor fluorophores and PDA. In particular, we show the sensing of a model protein molecule streptavidin. We also demonstrate the covalent-binding of bovine serum albumin (BSA) to the liposome surface with FRET mechanism. These interactions between the bilayer liposomes and protein molecules can be sensed in real-time. The proposed method is a general method for sensing small chemical and large biochemical molecules. Since fluorescence is intrinsically more sensitive than colorimetry, the detection limit of the assay can be in sub-nanomolar range or lower8. Further, PDA can act as a universal acceptor in FRET, which means that multiple sensors can be developed with PDA (acceptor) functionalized with donors and different receptors attached on the surface of PDA liposomes.

We demonstrate FRET between conjugated polymer polydiacetylene (PDA) and fluorophore attached to the surface of PDA liposomes for the sensing of biomolecules. PDA liposomes also contained receptor molecules on their surfaces for biomolecules to be used as probes. Ligand-receptor interactions lead to changes in the FRET efficiency between the fluorophore and PDA which is the basis of the sensing mechanism.

Echtzeit-Überwachung von Ligand-Rezeptor-Wechselwirkungen mit Fluorescence Resonance Energy Transfer

FRET is a process  whereby energy is non-radiatively transferred from an excited donor molecule to  a ground-state acceptor molecule through long-range ...

Biologie

Development of a Colloidal Gold-based Immunochromatographic Test Strip for Detection of Cetacean Myoglobin

This protocol describes the development of a colloidal gold immunochromatographic test strip based on the sandwich format that can be used to differentiate the myoglobin (Mb) of cetaceans from that of seals and other animals. The strip provides rapid and on-the-spot screening for cetacean meat, thereby restraining its illegal trade and consumption. Two monoclonal antibodies (mAbs) with reactivity toward the Mb of cetaceans were developed. The amino acid sequences of Mb antigenic reactive regions from various animals were analyzed in order to design two synthetic peptides (a general peptide and a specific peptide) and thereafter raise the mAbs (subclass IgG1). The mAbs were selected from hybridomas screened by indirect ELISA, western blot and dot blot. CGF5H9 was specific to the Mbs of rabbits, dogs, pigs, cows, goats, and cetaceans while it showed weak to no affinity to the Mbs of chickens, tuna and seals. CSF1H13 can bind seals and cetaceans with strong affinity but showed no affinity to other animals. Cetacean samples from four families (Balaenopteridae, Delphinidae, Phocoenidae and Kogiidae) were used in this study, and the results indicated that these two mAbs have broad binding ability to Mbs from different cetaceans. These mAbs were applied on a sandwich-type colloidal gold immunochromatographic test strip. CGF5H9, which recognizes many species, was colloid gold-labeled and used as the detection antibody. CSF1H13, which was coated on the test zone, detected the presence of cetacean and seal Mbs. Muscle samples from tuna, chicken, seal, five species of terrestrial mammals and 15 species of cetaceans were tested in triplicate. All cetacean samples showed positive results and all the other samples showed negative results.

This protocol describes the development of two IgG class monoclonal antibodies (mAbs) strongly reactive to myoglobin of cetaceans. These mAbs are applied on a colloidal gold immunochromatographic test strip based on the sandwich format to differentiate the Mb of cetaceans from seal and other animals.