Name: fluorescence-based monitoring of pad4 activity via a pro-fluorescence substrate analog
Uploaded: 2014-11-05T13:00:00.000+00:00
Duration: 8 min 37 s
Description: Watch this Scientific Journal Video about Fluorescence-based Monitoring of PAD4 Activity via a Pro-fluorescence Substrate Analog at JoVE.com

This work was supported by the Lehigh University Start-up Package. We thank Dr. Walter Fast for providing the GST-PAD4 plasmid for protein expression.

1. PAD4 Transformación, Expresión y Purificación <ol><li> Para PAD4 Transformación, transformar el plásmido pGEX que contiene GST fusión PAD4 en E. químicamente competente Coli (BL21 (DE3)) las células para la expresión de proteínas utilizando el siguiente procedimiento. Preparar (cloruro de calcio) químicamente competente E. Células de E. coli (BL21 (DE3)) de acuerdo con protocolos estándar. <ol><li> Descongelar 50 l de preparado previamente químicamente competentes BL21 (DE3) células en hielo y se mezcla con 1 l de la plásmido pGEX que contiene el gen PAD4 en un tubo de cultivo de 5 ml. Incubar la mezcla en hielo durante 10 min mientras se agitaba suavemente cada 2 min. </li><li> Choque térmico de las células mediante la colocación de la mezcla en un baño de agua de 42 ° C durante 40 segundos. Inmediatamente colocar la mezcla de células-plásmido de nuevo en hielo durante 2 min para permitir que las células se recuperen. Añadir 1 ml de caldo LB estéril a la mezcla y colocar en hielo durante 1 min. </li><li> Incubar el calor conmocionado células a 37 ° C, agitando a 250 rpm durante 1hora Pipetear 75 l de las células transformadas en una placa de agar resistente a la ampicilina y se incuba a 37 ° C durante 15 hr. Placa Almacenar a 4 ° C. </li></ol></li><li> Expresión PAD4. <ol><li> Escoja 1 colonia de células BL21 (DE3) de la placa de agar ampicilina y lugar en 5 ml de LB que contienen ampicilina 1x. Colocar en incubadora y agitar O / N a 37 ° C. </li><li> La transferencia de los 5 ml de LB (arranque) en 1 L de LB estéril que contiene trihidrato de ampicilina 1x (MW 403.45 g / mol). Place crecimiento en un incubador con agitación 37 ° C. Supervisar el OD 600 del crecimiento. Cuando el crecimiento llega a un OD 600 de 0,3, mueva el crecimiento en 16 ° C agitando incubadora. </li><li> Al llegar a un OD 600 de 0,6, inducir a las células con isopropylthiagalactoside 0,3 mM (IPTG, MW 238.30 g / mol). Permitir que las células se dan durante 15 horas a 16 ° C. </li><li> Las células de la cosecha por centrifugación a 4000 xg durante 20 min a 0 ° C. Retirar el sobrenadante y tienda de pellets a -80 ° C. </li></ol></li><li> PAD4 Purificación <ol><li> Vuelva a suspender el sedimento que contiene el PAD4 expresado en BL21 (DE3) las células en un tampón de 50 mM de NaCl (PM 58,44 g / mol), 300 mM NaH 2 PO 4 (PM 119,98 g / mol), 10 mM de imidazol (MW 68.077 g / mol), 0,1 mM de fluoruro de fenilmetilsulfonilo (PMSF, MW 174.94 g / mol) y ditiotreitol 1 mM (DTT, 154,25 g / mol), pH = 8,0. </li><li> Lisar las células a través de sonicación durante 15 min a 4 ° C. Después de la sonicación, centrifugar el lisado celular a 20.000 xg durante 20 min a 0 ° C. Retirar y guardar el sobrenadante. Lotes del sobrenadante con el glutatión (GSH) perlas de agarosa durante 30 min a RT. </li><li> Escurrir sobrenadante de GST cuentas / columna a través de la gravedad. Lavar los granos con 4 x 25 ml de 1x PBS (solución salina tamponada con fosfato, pH = 8,0) a TA. </li><li> Eluir PAD4 con 2 x 10 ml de tampón de elución, tris 50 mM (hidroximetil) aminometano (Tris Base, MW 121.14 g / mol), glutatión 10 mM (GSH, MW 307.32 g / mol), pH = 8,0. </li><li> Concéntrese PAD4 usando 100k MW corte-otubos de centrífuga ff y centrifugar a 4000 xg durante 20 min a 4 ° C. Alícuota de proteínas en 200 l volúmenes de 1,0 ml tubos de microcentrífuga y se almacena a -80 ° C. </li></ol></li></ol> 2. Preparación de las soluciones de archivo para tampones <ol><li> Pesar de cloruro de sodio (NaCl, MW 58,44 g / mol) y preparar una solución 2 M. Mezcle la solución hasta que se aclare. </li><li> Pesar Tris (hidroximetil) aminometano (Tris Base, MW 121.14 g / mol) y preparar una solución 2 M, pH = 8,0. Mezcle la solución hasta que se aclare. </li><li> Pesar dihidrato de cloruro de calcio (CaCl 2 2H 2 O, MW 147.01 g / mol) y preparar una solución de 500 mM. Mezcle la solución hasta que se aclare. </li><li> Pesar Tris (2-carboxietil) fosfina (TCEP, MW 250.19 g / mol) y preparar una solución de 200 mM. Mezcle la solución hasta que se aclare y se almacena a -20 ° C. </li><li> Pesar ditiotreitol (DTT, PM 154,25 g / mol) y preparar una solución de 1 M. Mezcle la solución hasta que se aclare y se almacena a -20 ° C. </li><li> Prepararuna solución al 0,5% de Triton X-100. </li><li> Pesar el ácido etilendiaminotetraacético (EDTA, MW 292.24 g / mol) y preparar una solución de 100 mM. Mezcle la solución hasta que se aclare. </li><li> Pesar Z- Arg Arg 7- amido 4- metilcumarina clorhidrato (ZRcoum, MW 621.69 g / mol) y preparar una solución 10 mM en dimetilsulfóxido (DMSO). </li></ol> 3. Ensayo PAD4 a 37 ° C <ol><li> Del 10 mM ZRcoum acciones, preparar una solución de 125 mM de ZRcoum en agua. Esta solución 125 mM ZRcoum será Solución A. </li><li> Preparar un tampón de NaCl 62,5 mM, 62,5 mM Tris, 12.5 mM CaCl 2, DTT 6,25 mM, y 5 PAD4 M (pH = 8,0). Esta será la solución B. </li><li> Preparar un tampón de NaCl 62,5 mM, 62,5 mM Tris, 12,5 mM CaCl 2, y 6,25 mM TDT (pH = 8,0). Esta será la solución C. </li><li> Pesar tripsina, cristalino (de páncreas bovino) y preparar una 10 mg / ml en EDTA 100 mM. Mezcle la solución hasta que se aclare y almacenar a -20° C. Este será Solución D. </li><li> Obtener una placa de 96 pocillos y designar un número de pozos para los controles (sin PAD4) y ​​pocillos de ensayo (+ PAD4). </li><li> Pipetear 160 l de solución B en los pocillos de ensayo y pipeta de 160 l de solución C en los pocillos de control. Incubar durante 15 minutos a 37 ° C. </li><li> Pipetear 40 l de la solución A en todos los pocillos y se incuba a 37 ° C durante 45 min. </li><li> Pipeta de 10 l de dH 2 O a la mitad de los pozos de control y la mitad de los pozos de prueba. Para la otra mitad de los pocillos de control y los controles de ensayo, pipetas de 10 l de solución de D. Incubar durante 10 minutos a 37 ° C. </li><li> Obtener una lectura de fluorescencia en un lector de multimodal con una longitud de onda de excitación de 345 nm y una longitud de onda de emisión de 465 nm. </li></ol> 4. Ensayo PAD4 a TA <ol><li> Del 10 mM ZRcoum acciones, preparar una solución de 50 mM de ZRcoum en agua. Esta solución ZRcoum 50 M será Solución A. </li><li> Preparar un tampón de Na 100 mMCl, 100 mM Tris, 20 mM CaCl 2, 2 mM TCEP, 0,02% de Triton X-100, y 8 PAD4 M (pH = 8,0). Esta será la solución B. </li><li> Preparar un tampón de NaCl 100 mM, Tris 100 mM, 20 mM CaCl 2, 2 mM TCEP, y 0,02% de Triton X-100 (pH = 8,0). Esta será la solución C. </li><li> Pesar tripsina, cristalino (de páncreas bovino) y preparar una 10 mg / ml en EDTA 100 mM. Mezcle la solución hasta que se aclare y se almacena a -20 ° C. Este será Solución D. </li><li> Obtener una placa de 384 pocillos y designar pozos para los controles (sin PAD4) y ​​pozos de prueba (+ PAD4). </li><li> Pipetear 25 l de solución B en los pocillos de ensayo y pipeta de 25 l de solución C en los pocillos de control. Permitir tampón con / sin PAD4 incubar en los pocillos durante 20 min a RT. </li><li> Pipeta 25 l de solución C en todos los pocillos e incubar durante 45 minutos a temperatura ambiente. </li><li> Pipeta de 10 l de dH 2 O a la mitad de los pozos de control y la mitad de los pozos de prueba. Para la otra mitad de los pocillos de control y controles de prueba, pipeteee 10 l de solución de D. Incubar a TA durante 10 min. </li><li> Obtener una lectura de fluorescencia en un lector de multimodal con una longitud de onda de excitación de 345 nm y una longitud de onda de emisión de 465 nm. </li></ol> 5. PAD4 Tiempo Ensayos del curso a 37 ° C y RT <ol><li> Del 10 mM ZRcoum acciones, preparar una solución de 125 mM de ZRcoum en agua. Esta solución 125 mM ZRcoum será Solución A. </li><li> Preparar un tampón de NaCl 62,5 mM, 62,5 mM Tris, 12.5 mM CaCl 2, DTT 6,25 mM, y 5 PAD4 M (pH = 8,0). Esta será la solución B. </li><li> Pesar tripsina, cristalino (de páncreas bovino) y preparar una 10 mg / ml en EDTA 100 mM. Mezcle la solución hasta que se aclare y se almacena a -20 ° C. Esta será la solución C. </li><li> Obtener 2 placas de 96 pocillos y pipeta de 160 l de solución B en # cantidad de pozos (# de pozos dependerá de cuántos puntos de tiempo). </li><li> Incubar una placa de 96 pocillos a 37 ° C. Incubar la otra placa de 96 pocillos a TA. </li><li> Pepitaette 40 l de la solución A en todos los pocillos en ambas placas de 96 pocillos e inmediatamente pipeta de 10 l de solución de C en los pocillos t = 0 min. Continuar la adición de 10 l de solución de C en los puntos de tiempo específicos hasta que todos los pozos han sido templado. </li><li> Obtener una lectura de fluorescencia en un lector de multimodal con una longitud de onda de excitación de 345 nm y una longitud de onda de emisión de 465 nm. </li></ol> 6. PAD4 ensayo de inhibición a RT <ol><li> Del 10 mM ZRcoum acciones, preparar una solución de 83 mM de ZRcoum en agua. Esta solución ZRcoum 83 M será Solución A. </li><li> Pesar Cl-amidina (MW 424.8 g / mol) y preparar una solución madre 100 mM. Esta será la solución B. </li><li> Preparar un tampón de NaCl 100 mM, Tris 100 mM, 20 mM CaCl 2, 2 mM TCEP, 0,02% de Triton X-100, y 8 M PAD4 (pH = 8,0). Esta será la solución C. </li><li> Preparar un tampón de NaCl 100 mM, Tris 100 mM, 20 mM CaCl 2, 2 mM TCEP, y 0,02% de Triton X-100 (pH = 8,0). Thes que será la solución D. </li><li> Pesar tripsina, cristalino (de páncreas bovino) y preparar una 10 mg / ml en EDTA 100 mM. Mezcle la solución hasta que se aclare y se almacena a -20 ° C. Este será Solución E. </li><li> Obtener una placa de 384 pocillos y designar pozos para los controles (sin PAD4), pozos de prueba (+ PAD4), y pozos de inhibición (PAD4 + Cl-amidina). </li><li> Pipetear 25 l de solución C en los pocillos de ensayo y pipeta de 25 l de solución D en los pocillos de control. Permitir tampón con / sin PAD4 incubar en los pocillos durante 20 min a RT. </li><li> Pipeta de 10 l de solución B en pozos de inhibición y se incuban durante 20 minutos a Rte. </li><li> Pipeta 15 l de solución A en todos los pocillos e incubar durante 45 minutos a temperatura ambiente. </li><li> Pipetear 10 l de solución E a todos los pocillos. Incubar a TA durante 10 min. </li><li> Obtener una lectura de fluorescencia en un lector de multimodal con una longitud de onda de excitación de 345 nm y una longitud de onda de emisión de 465 nm. </li></ol> 7. Ensayo de lisado celular brutoa TA <ol><li> Después de cosechar las células de crecimiento PAD4 1 L, Resuspender las células en 10 ml de Tris 50 mM, NaCl 50 mM, 0,1 mM de fluoruro de fenilmetilsulfonilo (PMSF, MW 174.94 g / mol) y ditiotreitol 1 mM (DTT, 154,25 g / mol), pH = 8,0. </li><li> Lisar las células a través de sonicación durante 15 min a 4 ° C. Se centrifuga el lisado celular a 20.000 xg durante 20 min a 0 ° C. Se concentra el sobrenadante claro a la mitad del volumen mediante centrifugación a 4000 rpm a 4 ° C utilizando 100k MW tubos de centrífuga de corte. </li><li> Del 10 mM ZRcoum acciones, preparar una solución de 250 mM de ZRcoum en agua. Esta solución 250 mM ZRcoum será Solución A. </li><li> Preparar un tampón de NaCl 100 mM, Tris 100 mM, 20 mM CaCl 2, y 2 mM TCEP (pH = 8,0). Esta será la solución B. </li><li> Pesar tripsina, cristalino (de páncreas bovino) y preparar una 10 mg / ml en EDTA 100 mM. Mezcle la solución hasta que se aclare y se almacena a -20 ° C. Esta será la solución C. </li><li> Obtener una placa de 384 pocillos negro y diseñocomió pozos para los controles (sin lisado celular) y pocillos de ensayo (+ lisado celular). </li><li> Pipetear 100 l de solución B en todos los pozos y las pipetas de 60 l de lisado celular en los pocillos de ensayo. Incubar durante 20 min a TA. </li><li> Pipeta 40 l de solución A en todos los pocillos e incubar durante 45 minutos a temperatura ambiente. </li><li> Pipetear 10 l de la solución C a todos los pocillos. Incubar a TA durante 10 min. </li><li> Obtener una lectura de fluorescencia en el lector multimodal con una longitud de onda de excitación de 345 nm y una longitud de onda de emisión de 465 nm. </li></ol>

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Devel. 12, 616-627 (2009).</li><li>Li, P., 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=Regulation+of+p53+target+gene+expression+by+peptidylarginine+deiminase+4.">Regulation of p53 target gene expression by peptidylarginine deiminase 4.</a> Mol. Cell Biol. 28, 4745-4758 (2008).</li><li>Wang, 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=Anticancer+peptidylarginine+deiminase+(PAD)+inhibitors+regulate+the+autophagy+flux+and+the+mammalian+target+of+rapamycin+complex+1+activity.">Anticancer peptidylarginine deiminase (PAD) inhibitors regulate the autophagy flux and the mammalian target of rapamycin complex 1 activity.</a> J. Biol. Chem. 287, 25941-25953 (2012).</li><li>Slack, J. L., Causey, C. P., Thompson, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+arginine+deiminase+4:+a+target+for+an+epigenetic+cancer+therapy.">Protein arginine deiminase 4: a target for an epigenetic cancer therapy.</a> Cell. Mol. Life Sci. 68, 709-720 (2011).</li><li>Luo, Y., Knuckley, B., Lee, Y. H., Stallcup, M. R., Thompson, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fluoroacetamidine-based+inactivator+of+protein+arginine+deiminase+4:+design,+synthesis,+and+in+vitro+and+in+vivo+evaluation.">A fluoroacetamidine-based inactivator of protein arginine deiminase 4: design, synthesis, and in vitro and in vivo evaluation.</a> J. Am. Chem. Soc. 128, 1092-1093 (2006).</li><li>Knuckley, B., 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=Substrate+specificity+and+kinetic+studies+of+PADs.">Substrate specificity and kinetic studies of PADs.</a> Biochemistry. 1, 4852-4863 (2010).</li><li>Knipp, M., Vasak, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+colorimetric+96-well+microtiter+plate+assay+for+the+determination+of+enzymatically+formed+citrulline.">A colorimetric 96-well microtiter plate assay for the determination of enzymatically formed citrulline.</a> Anal. Biochem. 286, 257-264 (2000).</li><li>Knuckley, B., 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+fluopol-ABPP+HTS+assay+to+identify+PAD+inhibitors.">A fluopol-ABPP HTS assay to identify PAD inhibitors.</a> Chem. Commun. 46, 7175-7177 (2010).</li><li>Bicker, K. L., Subramanian, V., Chumanevich, A. A., Hofseth, L. J., Thompson, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seeing+citrulline:+development+of+a+phenylglyoxal-based+probe+to+visualize+protein+citrullination.">Seeing citrulline: development of a phenylglyoxal-based probe to visualize protein citrullination.</a> J. Am. Chem. Soc. 134, 17015-17018 (2012).</li><li>Wang, Q., Priestman, M. A., Lawrence, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+protein+arginine+deiminase+activity+by+using+fluorescence+quenching:+multicolor+visualization+of+citrullination.">Monitoring of protein arginine deiminase activity by using fluorescence quenching: multicolor visualization of citrullination.</a> Angew. Chem. Int. Ed. Engl. 52, 2323-2325 (2013).</li><li>Jones, J. E., 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=Synthesis+and+screening+of+a+haloacetamidine+containing+library+to+identify+PAD4+selective+inhibitors.">Synthesis and screening of a haloacetamidine containing library to identify PAD4 selective inhibitors.</a> ACS Chem. Biol. 7, 160-165 (2012).</li><li>Dreyton, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Optimization and characterization of a pan protein arginine deiminase (PAD) inhibitor. , (2010).</li><li>Shimoyama, 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=Deimination+stabilizes+histone+H2A/H2B+dimers+as+revealed+by+electrospray+ionization+mass+spectrometry.">Deimination stabilizes histone H2A/H2B dimers as revealed by electrospray ionization mass spectrometry.</a> J. Mass Spectrom. 45, 900-908 (2010).</li><li>Luo, 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=Inhibitors+and+inactivators+of+protein+arginine+deiminase+4:+functional+and+structural+characterization.">Inhibitors and inactivators of protein arginine deiminase 4: functional and structural characterization.</a> Biochemistry. 45, 11727-11736 (2006).</li></ol>

The authors have nothing to disclose.

Herein, a fluorescence based assay was successfully developed that monitors the activity of PAD4. The assay has proven to be incredibly robust in a number of high-throughput conditions and is also compatible with whole cell lysates37.
The affinity between the histone proteins and the DNA strands surrounding it loosens due the neutralization of the positive charge on the arginine side chain upon citrullination. It was envisioned that the neutralization of the arginine side-chain coul...

Un gran número de proteínas de mamíferos son muy modificada por la acción de enzimas después de la biosíntesis de proteínas por el ribosoma. Estas modificaciones post-traduccionales (PTMs) pueden aumentar en gran medida la diversidad funcional del proteoma cambiando el tamaño, carga, estructura y estado de oligomerización (entre otras características) de las proteínas 1-3. Como resultado, el cambio en la estructura de la proteína puede conducir a consecuencias fisiológicas, tales como la degradación de proteínas, la diferenciación celular, la señalización, la modulación en la expresión génica, y las interacciones proteína-proteína. Mientras que estas modificaciones son frecuentes en un gran porcentaje de todas las proteínas humanas, los extremos terminales de las proteínas histonas se someten a un número inusualmente alto de modificaciones covalentes 4. Las proteínas histonas son una familia de proteínas estructurales que facilitan la condensación del ADN genómico. Las modificaciones covalentes de las colas de las histonas no estructurados se llevan a cabo y regulados por una series de enzimas que pueden catalizar la modificación covalente de residuos (escritores), revertir las mismas modificaciones (borradores), y distinguir entre los cambios que se imprime en las colas de las histonas (lectores) 5-7. De hecho, la mayoría de los PTM conocidos se puede observar dentro de este segmento corto de la histona incluyendo la metilación, fosforilación, acetilación, sumoylation, ubiquitinación, y citrullination 8. Citrullination implica la conversión de peptidil-arginina a la no t RNA peptidil-citrulina codificada (Figura 1A). Las proteínas, responsables de esta cadena de neutralización lado, son miembros de la PAD (p eptide un d eiminase rginine) familia de proteínas, todos los cuales son enzimas dependientes de calcio. 9,10. Hasta la fecha, cinco miembros de la familia PAD se han descrito (PAD1, PAD2, PAD3, PAD4, y PAD6). Cada miembro de esta familia aparece a proteínas diana celulares distintas y también muestra uniperfiles de distribución en tejidos Que. PAD4 es el único miembro de esta familia de proteínas conocida por ser localizada dentro del núcleo a través de una secuencia de localización nuclear 11. Por consiguiente, se ha demostrado que deiminate una serie de objetivos nucleares, incluyendo cadenas laterales de arginina en las colas N-terminales de las histonas H2A residuo de arginina 3 (H2R3), H3 (H3R2, H3R17, y H3R26) y H4 (H4R3) 12, 13. Mientras que cada una de las isoenzimas PAD tienen funciones fisiológicas específicas y críticas, PAD4 ha recibido mucha más atención debido a su papel en un número de procesos humanos en ambas células enfermas y sanas. Recientemente, PAD4 se demostró que era un miembro de la red transcripcional pluripotencia 14. Ambos niveles de expresión y actividad PAD4, se mostró a ser elevados durante la reprogramación y en estado fundamental estados pluripotentes en ratones. Mediante el control de la regulación de los genes de células madre, PAD4 puede retener un papel fundamental en la eficiencia de la reprogramación celular. PAD4 también ha sido implicado en laformación de trampas extracelulares de neutrófilos, que tras la unión de patógenos permitir su depuración de sistema. El hypercitrullination de proteínas histonas por PAD4 induce descondensación de la cromatina, que sirve como material de base para la encapsulación de bacterias patógenas por la trampa extracelular, por lo tanto alejar las infecciones bacterianas 15,16. Además, PAD4 se ha encontrado para desempeñar un papel activo en un número de enfermedades humanas. Se ha demostrado previamente que la expresión aberrante de PAD4 se asocia con la aparición y severidad de la artritis reumatoide, la enfermedad de Alzheimer, enfermedad de Parkinson, y esclerosis múltiple 17. De hecho, la presencia de anticuerpos anti-proteína citrulinados es uno de los biomarcadores diagnósticos y pronósticos más confiables y definitivas de la artritis reumatoide 18. Asimismo, la actividad PAD4 dysregulated recientemente se ha observado en un número de cánceres humanos incluyendo ovario, mama, pulmón, unad cánceres esofágicos 19-22. El vínculo entre PAD4 y el cáncer se ha demostrado que ser mediada a través de la oncogén ELK1 o a través de la proteína supresora de tumores p53 22,23 y el trabajo previo ha sugerido que PAD4 podría ser un anti-cáncer de nueva diana terapéutica 17,24,25. Como prueba de principio estudio, el agotamiento de los PAD4 través shRNA en la línea celular de cáncer colorrectal HCT116 fue revelado a ser suficiente para inducir la apoptosis y la detención del ciclo celular 26. Un inhibidor irreversible PAD4 desarrollado recientemente condujo a una reducción del setenta por ciento de la masa tumoral en ratones 27. Muy notablemente, la inhibición PAD4 parecía actuar como una terapia dirigida que resultó en la destrucción selectiva de las células cancerosas sin dañar las células no transformadas. El uso de moléculas pequeñas para desactivar la función de PAD4 puede llegar a ser una nueva y poderosa estrategia para dirigirse a las células cancerosas o para aumentar quimioterapéuticos de cáncer existentes 28. Por desgracia, un pocarpa inhibidor reversible PAD4 aún no se ha descubierto. Una serie de inhibidores covalentes se han desarrollado utilizando la / mango imidina flúor cloro que imita el sustrato arginina 27,29,30 y han demostrado ser herramientas prácticas para comprender el papel de PAD4 tanto en las células sanas y enfermas estatales. Sin embargo, estas moléculas inhiben todos los PAD activos con potencia similar. Por lo tanto, la necesidad de un ensayo fácil de que informa sobre la actividad de PAD4 es crucial. Hasta la fecha, PAD4 ensayos se han descrito que enlazan la liberación de amoníaco a partir de la reacción a una lectura colorimétrica 31, utilizar un análogo de sustrato chloroamidine marcado con fluorescencia para el ensayo de polarización de fluorescencia 32, se basan en la reacción de ácido asistida entre glioxal y citrulina 33, y par la actividad PAD4 a una fluorescencia desatenuación paso 34. De estos, sólo la estrategia haloacetamidine modificador covalente ha demostrado ser compatible con scre de alto rendimientoplataformas Ening 32,35,36. Se describe un ensayo basado en la fluorescencia superficial que mide de forma fiable la actividad de PAD4. El ensayo, que muestra una fuerte relación señal-ruido, la velocidad de análisis, y la robustez de la medición, tiene el potencial de descubrir un inhibidor de PAD4 verdaderamente potente y selectivo.

<table><tbody><tr><td>LB Broth, Miller (LURIA-BERTANI)</td><td>Amresco</td><td>J106-2KG</td><td>&lt;a href=&quot;http://www.amresco-inc.com/&quot;&gt;http://www.amresco-inc.com&lt;/a&gt;</td></tr><tr><td>Ampicillin Trihydrate (Off-white Powder), Fisher BioReagents</td><td>Fisher Scientific</td><td>BP902-25&amp;nbsp;</td><td>&lt;a href=&quot;http://www.fishersci.com/&quot;&gt;http://www.fishersci.com&lt;/a&gt;</td></tr><tr><td>Isopropylthiagalactonse (IPTG)</td><td>Life Technogolies</td><td>15529-019</td><td>&lt;a href=&quot;http://www.lifetechnologies.com/&quot;&gt;http://www.lifetechnologies.com&lt;/a&gt;</td></tr><tr><td>All Buffer Salts&amp;nbsp;</td><td>Fisher Scientific</td><td></td><td>Can purchase from Fisher Scieintifc; VWR; Acros; Sigma-Aldrich; etc.</td></tr><tr><td>Protino Glutathione (GSH) agarose&amp;nbsp;</td><td>Machery-Nagel</td><td>745500.10</td><td>Store at 4 &amp;deg;C; http://www.mn-net.com/</td></tr><tr><td>Chloroamidine&amp;nbsp;</td><td>Cayman Chemical&amp;nbsp;</td><td>10599</td><td>Store at -20 &amp;deg;C; https://www.caymanchem.com/app/template/Home.vm</td></tr><tr><td>Triton X-100</td><td>Sigma Aldrich</td><td>X100-100ML</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/&quot;&gt;www.sigmaaldrich.com&lt;/a&gt;</td></tr><tr><td>Corning/Nunclon MicroWell plates (96 and 384)</td><td></td><td></td><td>Purchase from Corning; Sigma-Aldrich</td></tr><tr><td>Tecan Infinite 200 / Tecan i-control microplate reader software</td><td></td><td></td><td>&lt;a href=&quot;http://www.tecan.com/&quot;&gt;www.tecan.com&lt;/a&gt;</td></tr><tr><td>Europium Ex. 340/40 Em. 475/15 filter</td><td></td><td></td><td>&lt;a href=&quot;http://www.perkinelmer.com/&quot;&gt;http://www.perkinelmer.com&lt;/a&gt;</td></tr></tbody></table>

fluorescence-based monitoring of pad4 activity via a pro-fluorescence substrate analog

Post-translational modifications may lead to altered protein functional states by increasing the covalent variations on the side chains of many protein substrates. The histone tails represent one of the most heavily modified stretches within all human proteins. Peptidyl-arginine deiminase 4 (PAD4) has been shown to convert arginine residues into the non-genetically encoded citrulline residue. Few assays described to date have been operationally facile with satisfactory sensitivity. Thus, the lack of adequate assays has likely contributed to the absence of potent non-covalent PAD4 inhibitors. Herein a novel fluorescence-based assay that allows for the monitoring of PAD4 activity is described. A pro-fluorescent substrate analog was designed to link PAD4 enzymatic activity to fluorescence liberation upon the addition of the protease trypsin. It was shown that the assay is compatible with high-throughput screening conditions and has a strong signal-to-noise ratio. Furthermore, the assay can also be performed with crude cell lysates containing over-expressed PAD4.

Inicialmente, se demostró que ZRcoum puede informar sobre la actividad de PAD4 en un formato de placa de 96 pocillos. Los pocillos se incubaron con el sustrato ZRcoum y en presencia / ausencia de PAD4. Tras un período de incubación de 45 min a 37 ° C, la fluorescencia se midió con un 340/40 - 475/15 nm filtro (Figura 2A). Como era de esperar, los niveles de fluorescencia se mantuvo bajo como fluoróforo en ZRcoum (o el citrulinado ZRcoum)</...

Watch this Scientific Journal Video about Fluorescence-based Monitoring of PAD4 Activity via a Pro-fluorescence Substrate Analog at JoVE.com

Fluorescence-based Monitoring of PAD4 Activity via a Pro-fluorescence Substrate Analog

PAD4 is an enzyme responsible for the conversion of peptidyl-arginine to peptidyl-citrulline. Dysregulation of PAD4 has been implicated in a number of human diseases. A facile and high-throughput compatible fluorescence based PAD4 assay is described.

Monitoreo basado en la fluorescencia de PAD4 actividad a través de un sustrato Analog Pro-fluorescencia

Post-translational modifications may lead to altered protein functional states by increasing the covalent variations on the side chains of many protein ...

fluorescence-based-monitoring-pad4-activity-via-pro-fluorescence

Research

JoVE Journal

Chemistry

9.8K vistas.  Lehigh University. PAD4 is an enzyme responsible for the conversion of peptidyl-arginine to peptidyl-citrulline. Dysregulation of PAD4 has been implicated in a number of human diseases. A facile and high-throughput compatible fluorescence based PAD4 assay is described.

Video: Fluorescence-based Monitoring of PAD4 Activity via a Pro-fluorescence Substrate Analog

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

Métodos de detección de fluorescencia para las plataformas de las gotas de microfluidos

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

Investigación

Bioingeniería

An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation

Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to measure protein phosphorylation cannot preserve the heterogeneity in phosphorylation across individual proteins. The single-molecule pull-down (SiMPull) assay was developed to investigate the composition of macromolecular complexes via immunoprecipitation of proteins on a glass coverslip followed by single-molecule imaging. The current technique is an adaptation of SiMPull that provides robust quantification of the phosphorylation state of full-length membrane receptors at the single-molecule level. Imaging thousands of individual receptors in this way allows for quantifying protein phosphorylation patterns. The present protocol details the optimized SiMPull procedure, from sample preparation to imaging. Optimization of glass preparation and antibody fixation protocols further enhances data quality. The current protocol provides code for the single-molecule data analysis that calculates the fraction of receptors phosphorylated within a sample. While this work focuses on phosphorylation of the epidermal growth factor receptor (EGFR), the protocol can be generalized to other membrane receptors and cytosolic signaling molecules.

The present protocol describes sample preparation and data analysis to quantify protein phosphorylation using an improved single-molecule pull-down (SiMPull) assay.

Un ensayo optimizado de extracción de una sola molécula para la cuantificación de la fosforilación de proteínas

Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to ...

Bioquímica

Análisis de AMPc con cADDis: cribado de una sola célula a de alto rendimiento

Real-Time cAMP Dynamics in Live Cells Using the Fluorescent cAMP Difference Detector In Situ

cAMP Difference Detector In Situ (cADDis) is a novel biosensor that allows for the continuous measurement of cAMP levels in living cells. The biosensor is created from a circularly permuted fluorescent protein linked to the hinge region of Epac2. This creates a single fluorophore biosensor that displays either increased or decreased fluorescence upon binding of cAMP. The biosensor exists in red and green upward versions, as well as green downward versions, and several red and green versions targeted to subcellular locations. To illustrate the effectiveness of the biosensor, the green downward version, which decreases in fluorescence upon cAMP binding, was used. Two protocols using this sensor are demonstrated: one utilizing a 96-well plate reading spectrophotometer&#160;compatible with high-throughput screening and another utilizing single-cell imaging on a fluorescent microscope. On the plate reader, HEK-293 cells cultured in 96-well plates were stimulated with 10 &#181;M forskolin or 10 nM isoproterenol, which induced rapid and large decreases in fluorescence in the green downward version. The biosensor was used to measure cAMP levels in individual human airway smooth muscle (HASM) cells monitored under a fluorescent microscope. The green downward biosensor displayed similar responses to populations of cells when stimulated with forskolin or isoproterenol. This single-cell assay allows visualization of the biosensor location at 20x and 40x magnification. Thus, this cAMP biosensor is sensitive and flexible, allowing real-time measurement of cAMP in both immortalized and primary cells, and with single cells or populations of cells. These attributes make cADDis a valuable tool for studying cAMP signaling dynamics in living cells.

Autor destacado: Avance de la detección de AMPc en tiempo real en células mediante el biosensor cADDis

The protocol presented in this study illustrates the effectiveness of the cAMP Difference Detector In Situ in measuring cAMP through two methods. One method utilizes a 96-well plate reading spectrophotometer with HEK-293 cells. The other method demonstrates individual HASM cells under a fluorescent microscope.

Dinámica de cAMP en tiempo real en células vivas utilizando el detector de diferencia de cAMP fluorescente in situ

cAMP Difference Detector In Situ (cADDis) is a novel biosensor that allows for the continuous measurement of cAMP levels in living cells. The biosensor is ...

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane proteins including ion channels2, ion pumps3, and transporters4. Recent developments have combined site-specific fluorophore labeling alongside TEVC to 
concurrently examine the conformational dynamics at specific residues and function of these proteins on the surface of single cells.
We will describe a method to study the conformational dynamics of membrane proteins by simultaneously monitoring fluorescence and current changes using voltage-clamp fluorometry. This approach can be used to examine the molecular motion of membrane proteins site-specifically following cysteine replacement and site-directed fluorophore labeling5,6. Furthermore, this method provides an approach to determine distance constraints between specific residues7,8. 
This is achieved by selectively attaching donor and acceptor fluorophores to two mutated cysteine residues of interest.
In brief, these experiments are performed following functional expression of the desired protein on the surface of Xenopus leavis oocytes. The large surface area of these oocytes enables facile functional measurements and a robust fluorescence signal5. It is also possible to readily change the extracellular conditions such as pH, ligand or cations/anions, which can provide further information on the mechanism of membrane proteins4. Finally, recent developments 
have also enabled the manipulation of select internal ions following co-expression with a second protein9.
Our protocol is described in multiple parts. First, cysteine scanning mutagenesis proceeded by fluorophore labeling is completed at residues located at the interface of the transmembrane and extracellular domains. Subsequent experiments are designed to identify residues which demonstrate large changes in fluorescence intensity (&lt;5%)3 upon a conformational change of the protein. Second, these changes in fluorescence intensity are compared to the kinetic parameters of 
the membrane protein in order to correlate the conformational dynamics to the function of the protein10. This enables a rigorous biophysical analysis of the molecular motion of the target protein. Lastly, two residues of the holoenzyme can be labeled with a donor and acceptor fluorophore in order to determine distance constraints using donor photodestruction methods. It is also possible to monitor the relative movement of protein subunits following labeling with a donor and acceptor fluorophore.

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

We will describe a method which measures the kinetics of ion transport of membrane proteins alongside site-specific analysis of conformational changes using fluorescence on single cells. This technique is adaptable to ion channels, transporters and ion pumps and can be utilized to determine distance constraints between protein subunits.

Examinar la dinámica conformacional de las proteínas de membrana In situ Sitio-dirigida con fluorescencia de etiquetado

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane ...

Biología

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such probes typically consist of variants of CFP and YFP, intervened by a phosphorylatable sequence and a phospho-binding domain. Upon phosphorylation, the probe changes conformation, which results in a change of the distance or orientation between CFP and YFP, leading to a change in FRET efficiency (Fig 1). Several probes have been published during the last decade, monitoring the activity balance of multiple kinases and phosphatases, including reporters of PKA2, PKB3, PKC4, PKD5, ERK6, JNK7, Cdk18, Aurora B9 and Plk19. Given the modular design, additional probes are likely to emerge in the near future10. 
Progression through the cell cycle is affected by stress signaling pathways 11. Notably, the cell cycle is regulated differently during unperturbed growth compared to when cells are recovering from stress12.Time-lapse imaging of cells through the cell cycle therefore requires particular caution. This becomes a problem particularly when employing ratiometric imaging, since two images with a high signal to noise ratio are required to correctly interpret the results. Ratiometric FRET imaging of cell cycle dependent changes in kinase and phosphatase activities has predominately been restricted to sub-sections of the cell cycle8,9,13,14.
Here, we discuss a method to monitor FRET-based probes using ratiometric imaging throughout the human cell cycle. The method relies on equipment that is available to many researchers in life sciences and does not require expert knowledge of microscopy or image processing.

FRET-based reporters are increasingly used to monitor kinase and phosphatase activities in live cells. Here we describe a method on how to use FRET-based reporters to assess cell cycle-dependent changes in target phosphorylation.

Seguimiento de las actividades de quinasa y fosfatasa través del ciclo celular por Proporcional FRET

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such ...

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

F&ouml;rster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a &beta;-adrenergic receptor agonist and blocker.

F&#246;rster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

FRET Microscopía de Monitoreo en tiempo real de eventos de señalización en las células vivas Uso de biosensores unimoleculares

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or&#160;photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.

We demonstrate the use of fluorescence photo activation localization microscopy (FPALM) to simultaneously image multiple types of fluorescently labeled molecules&#160;within cells. The techniques described yield the localization of thousands to hundreds of thousands of individual fluorescent labeled proteins, with a precision of tens of nanometers&#160;within single cells.

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled ...

Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular organelles and those localized on the plasma membrane. Traditional fluorescence-based measurements lack the capability to distinguish membrane proteins residing in different organelles. Cutting edge methodologies transcend traditional methods by coupling pH-sensitive fluorophores with total internal reflection fluorescence microscopy (TIRFM). TIRF illumination excites the sample up to approximately 150 nm from the glass-sample interface, thus decreasing background, increasing the signal to noise ratio, and enhancing resolution. The excitation volume in TIRFM encompasses the plasma membrane and nearby organelles such as the peripheral ER. Superecliptic pHluorin (SEP) is a pH sensitive version of GFP. Genetically encoding SEP into the extracellular domain of a membrane protein of interest positions the fluorophore on the luminal side of the ER and in the extracellular region of the cell. SEP is fluorescent when the pH is greater than 6, but remains in an off state at lower pH values. Therefore, receptors tagged with SEP fluoresce when residing in the endoplasmic reticulum (ER) or upon insertion in the plasma membrane (PM) but not when confined to a trafficking vesicle or other organelles such as the Golgi. The extracellular pH can be adjusted to dictate the fluorescence of receptors on the plasma membrane. The difference in fluorescence between TIRF images at neutral and acidic extracellular pH for the same cell corresponds to a relative number of receptors on the plasma membrane. This allows a simultaneous measurement of intracellular and plasma membrane resident receptors. Single vesicle insertion events can also be measured when the extracellular pH is neutral, corresponding to a low pH trafficking vesicle fusing with the plasma membrane and transitioning into a fluorescent state. This versatile technique can be exploited to study localization, expression, and trafficking of membrane proteins.

Labeling the extracellular domain of a membrane protein with a pH sensitive fluorophore, superecliptic pHluorin (SEP), allows subcellular localization, expression, and trafficking to be determined. Imaging SEP-labeled proteins with total internal reflection fluorescence microscopy (TIRFM) enables the quantification of protein levels in the peripheral ER and plasma membrane.

La utilización de receptores de etiquetado pHluorin de Seguimiento de localización subcelular y el Tráfico

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular ...

Monitoreo basado en la fluorescencia de PAD4 actividad a través de un sustrato Analog Pro-fluorescencia

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Examinar la dinámica conformacional de las proteínas de membrana In situ Sitio-dirigida con fluorescencia de etiquetado

Seguimiento de las actividades de quinasa y fosfatasa través del ciclo celular por Proporcional FRET

FRET Microscopía de Monitoreo en tiempo real de eventos de señalización en las células vivas Uso de biosensores unimoleculares

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

La utilización de receptores de etiquetado pHluorin de Seguimiento de localización subcelular y el Tráfico