This work was supported by a grant from the Canadian Institutes for Health Research (MOP-86514).

Os procedimentos experimentais com camundongos foram aprovados pelo Comitê de Cuidados Animal da McGill University (protocolo 4966). 1. Preparação de extratos de proteínas a partir de células cultivadas <ol><li> Lavar as células cultivadas duas vezes com 10 ml de fosfato de gelo-solução salina tamponada (PBS). </li><li> Raspe células aderentes quer com um polícia de borracha ou um raspador de células de plástico em 1 ml de suspensão de PBS, arrefecido com gelo transferência para um tubo de microcentrífuga de 1,5 ml. </li><li> Centrifugação numa microcentrífuga durante 5 min a 700 xg, a 4 ° C. Aspirar PBS. </li><li> Adicionar 100 ul de tampão de lise arrefecido com gelo citoplasmática (Tabela 1) por 10 7 células, e pipetar para cima e para baixo. </li><li> Incubar em gelo durante 20 min. </li><li> Centrifugação durante 10 minutos à velocidade máxima numa microcentrífuga a 4 ° C. </li><li> Pellet Descartar. Transferir o sobrenadante para um novo tubo de microcentrífuga de 1,5 ml e manter em gelo. </li><li> Detarminho concentração de proteína (normalmente 1-10 mg / mL) utilizando o 23 ensaio de Bradford. </li><li> Alíquotas e armazenar extractos celulares a -80 ° C até à sua utilização. </li></ol> 2. Preparação de extratos de proteína de rato fígado e baço <ol><li> Euthanize um rato com inalação de CO 2. </li><li> Coloque o animal sacrificados em uma almofada limpo sobre uma placa de dissecação. Abra o abdômen com uma tesoura. </li><li> Dissecar o fígado e o baço, utilizando tesouras e pinças, e lave cada tecido em aproximadamente 50 ml de PBS gelado. </li><li> Tecidos em pequenos pedaços imediatamente cortada com um bisturi (por exemplo: cerca de 1 - 2 mm 3). </li><li> Sem demora, coloque pedaços de tecidos em uma nova cryotube e em seguida, encaixe congelá-los em nitrogênio líquido. Snap-congelados loja alíquotas de tecido a -80 ° C até à sua utilização. </li><li> Homogeneizar um pedaço de tecido congelado (cerca de 1 - 2 mm 3) em 0,25-0,5 ml degelo-tampão de lise frio citoplasmática (Tabela 1) com um homogeneizador de tecidos de 10 seg. </li><li> Transferir homogeneizado a 1,5 ml tubo de microcentrífuga e frio em gelo durante 20 min. </li><li> Centrifugação durante 10 minutos à velocidade máxima numa microcentrífuga a 4 ° C. </li><li> Descarte pellet e transferência sobrenadante para novo tubo de microcentrífuga de 1,5 ml. Mantenha no gelo. </li><li> Determinar a concentração de proteína (normalmente 1-10 mg / mL) utilizando o 23 ensaio de Bradford. </li><li> Alíquotas e armazenar extractos celulares a -80 ° C até à sua utilização. </li></ol> 3. Preparação de sonda radiomarcada-IRE <ol><li> Linearizar o plasmídeo contendo IRE-I-12.CAT 22 por incubação a 37 ° C durante 1 h com a endonuclease de restrição Xbal (1 U por ug de plasmídeo), que cliva a jusante da sequência IRE. O plasmídeo linearizado vai ser utilizado como molde para a transcrição in vitro. </li><li> Configurar uman na reacção de transcrição in vitro num volume total de 20 ul. Use as soluções stock mostrados na Tabela 2 e adicionar: 1 ul molde de plasmídeo linearizado, 4 ul de tampão de transcrição; 1 uL de mistura de mistura de ATP / CTP / GTP, 10 ul [α- 32 P] -UTP, 2 ul de ditiotreitol, 1 ul de inibidor de RNase e 1 ul de RNA-polimerase de T7. Misturar por pipetagem cima e para baixo. </li><li> Incubar a 40 ° C durante 1 h 24. </li></ol> 4. Purificação de IRE-sonda radiomarcada <ol><li> Terminar em reação de transcrição in vitro, adicionando 1 ml de 0,5 M EDTA, pH 8. Mix pipetando cima e para baixo. </li><li> Adicionar 10 ul de 10 mg / ml de ARNt, como transportador para uma melhor precipitação. Misturar por pipetagem cima e para baixo. </li><li> Adicionar 82.5 ul de acetato de amónio 3 M. Misture em vórtice. </li><li> Adicionar 273 ul de etanol. Misture em vórtice. </li><li> Deixar repousar à temperatura ambiente durante 5 min. </li><li> Girar durante 10 minutos à velocidade máxima numa microcentrífuga à temperatura ambiente. Sobrenadante Descartar. </li><li> Lave o peletizado com 100 ul de etanol a 70%. </li><li> Girar durante 10 minutos à velocidade máxima numa microcentrífuga à temperatura ambiente. Sobrenadante Descartar. </li><li> Ar sedimento seco durante 10 min. </li><li> Ressuspender o sedimento em 100 ul de duas vezes destilada, previamente autoclavada H 2 O. </li><li> Quantificar a radioactividade num contador de cintilação líquida, a sonda radiomarcada IRE alíquota e armazenar a -80 ° C até à sua utilização. Alíquotas congeladas podem ser utilizadas durante até 3 semanas. </li></ol> 5. Preparação de um gel de poliacrilamida nativa para EMSA <ol><li> Monte o gel (16 x 16 cm), usando 1,5 espaçadores mm e pente. </li><li> Para preparar um gel de poliacrilamida a 6% nativo, utilizar as soluções de estoque mostrados na Tabela 3 Misturar 7,5 ml de 40% de acrilamida:. Bisacrilamida, 5 ml de 5x TBE e 37,5 mldouble H2O destilada </li><li> Adicionar 0,5 ml de 10% de persulfato de amónio preparada recentemente (APS) e 25 ul tetrametiletilenodiamina (TEMED). </li><li> Derrame imediatamente a solução de acrilamida ao gel e deixá-lo polimerizar. Aguarde cerca de 30 min. </li><li> Montar o aparelho de eletroforese com gel, encher os tanques com 0,5x TBE e se conectar com a fonte de alimentação. </li></ol> 6. Electrophoretic Mobility Turno Assay <ol><li> Dilui-se 25 ug de extracto de proteína a partir de células ou tecidos com tampão de lise citoplasmática (Quadro 1) num volume total de 10 uL em (concentrações de proteína mais baixas podem também ser usados). Mantenha no gelo. Se necessário, adicionar 1 ml de 1: 4 diluído 2-mercaptoetanol (2-ME) para activar IRP1 dormente (concentração final: 2%) 25. </li><li> Dilui-se a sonda IRE radiolabeled em dobro H2O destilada para 200.000 cpm / mL, desnaturar calor em 95 °C durante 1 min, e arrefecer à temperatura ambiente durante pelo menos 5 min. </li><li> Configurar uma reação EMSA, adicionando 1 ml de sonda IRE radiolabeled ao extrato de proteína. </li><li> Incubar durante 20 minutos à temperatura ambiente. </li><li> Adicionar 1 ml de 50 mg / ml de heparina para a reacção (para inibir interacções de proteínas não-específicos com a sonda 9) e continuar a incubação durante mais 10 min. Alíquota da solução de reserva de heparina (50 mg / ml) e armazenar a -80 ° C. </li><li> Quando utilizando sondas radiomarcadas longas (&gt; 60 nucleótidos), adicionar 1 ul de ARNase T1 (1 U / ul) e incubar durante 10 min à temperatura ambiente para reduzir a proteína de ligação não específica com a sonda, e para permitir uma melhor separação do ARN / proteína complexo durante a electroforese. </li><li> Adicionar 3 ul de tampão de carga (glicerol a 80% + azul de bromofenol), misturar e carga sobre o não-desnaturante em gel de poliacrilamida a 6%. </li><li> Submeter o gel durante 60 minutos a 130 V (5 V / cm). </li><li> Transferência o gel para um grande papel de filtro e seco. </li><li> Expor a um filme e desenvolver auto-radiografia. O tempo de exposição pode variar desde 1 h (ou menos) até S / N. </li></ol>

<ol>
	<li>Fried, M., Crothers, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Equilibria+and+kinetics+of+lac+repressor-operator+interactions+by+polyacrylamide+gel+electrophoresis.">Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis.</a> Nucleic Acids Res. 9, 6505-6525 (1981).</li><li>Garner, M. M., Revzin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+gel+electrophoresis+method+for+quantifying+the+binding+of+proteins+to+specific+DNA+regions:+application+to+components+of+the+Escherichia+coli+lactose+operon+regulatory+system.">A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system.</a> Nucleic Acids Res. 9, 3047-3060 (1981).</li><li>Ryder, S. P., Recht, M. I., Williamson, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+protein-RNA+interactions+by+gel+mobility+shift.">Quantitative analysis of protein-RNA interactions by gel mobility shift.</a> Methods Mol Biol. 488, 99-115 (2008).</li><li>Hellman, L. M., Fried, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+mobility+shift+assay+(EMSA)+for+detecting+protein-nucleic+acid+interactions.">Electrophoretic mobility shift assay (EMSA) for detecting protein-nucleic acid interactions.</a> Nat Protoc. 2, 1849-1861 (2007).</li><li>Luscieti, 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=Novel+mutations+in+the+ferritin-L+iron-responsive+element+that+only+mildly+impair+IRP+binding+cause+hereditary+hyperferritinaemia+cataract+syndrome.">Novel mutations in the ferritin-L iron-responsive element that only mildly impair IRP binding cause hereditary hyperferritinaemia cataract syndrome.</a> Orphanet J Rare Dis. 8, 30 (2013).</li><li>Rio, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Filter-binding assay for analysis of RNA-protein interactions. 2012, 1078-1081 (2012).</li><li>Wang, 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=Iron-mediated+degradation+of+IRP2:+an+unexpected+pathway+involving+a+2-oxoglutarate-dependent+oxygenase+activity.">Iron-mediated degradation of IRP2: an unexpected pathway involving a 2-oxoglutarate-dependent oxygenase activity.</a> Mol. Cell. Biol. 24, 954-965 (2004).</li><li>Leibold, E. A., Munro, H. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoplasmic+protein+binds+in+vitro+to+a+highly+conserved+sequence+in+the+5'+untranslated+regions+of+ferritin+heavy-+and+light-subunit+mRNAs.">Cytoplasmic protein binds in vitro to a highly conserved sequence in the 5' untranslated regions of ferritin heavy- and light-subunit mRNAs.</a> Proc. Natl. Acad. Sci. USA. 85, 2171-2175 (1988).</li><li>Haile, D. J., Hentze, M. W., Rouault, T. A., Harford, J. B., Klausner, R. D. <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+interaction+of+the+iron-responsive+element+binding+protein+with+iron-responsive+RNA+elements.">Regulation of interaction of the iron-responsive element binding protein with iron-responsive RNA elements.</a> Mol. Cell. Biol. 9, 5055-5061 (1989).</li><li>Mueller, S., Pantopoulos, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+iron+regulatory+protein-1+(IRP1)+by+oxidative+stress.">Activation of iron regulatory protein-1 (IRP1) by oxidative stress.</a> Methods Enzymol. 348, 324-337 (2002).</li><li>Wang, J., Pantopoulos, K. <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+cellular+iron+metabolism.">Regulation of cellular iron metabolism.</a> Biochem J. 434, 365-381 (2011).</li><li>Hentze, M. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+the+iron-responsive+element+for+the+translational+regulation+of+human+ferritin+mRNA.">Identification of the iron-responsive element for the translational regulation of human ferritin mRNA.</a> Science. 238, 1570-1573 (1987).</li><li>Casey, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron-responsive+elements:+regulatory+RNA+sequences+that+control+mRNA+levels+and+translation.">Iron-responsive elements: regulatory RNA sequences that control mRNA levels and translation.</a> Science. 240, 924-928 (1988).</li><li>Dandekar, T., 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=Identification+of+a+novel+iron-responsive+element+in+murine+and+human+erythroid+d-aminolevulinic+acid+synthase+mRNA.">Identification of a novel iron-responsive element in murine and human erythroid d-aminolevulinic acid synthase mRNA.</a> EMBO J. 10, 1903-1909 (1991).</li><li>Gray, N. K., Pantopoulos, K., Dandekar, T., Ackrell, B. A. C., Hentze, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+regulation+of+mammalian+and+drosophila+citric+acid+cycle+enzymes+via+iron-responsive+elements.">Translational regulation of mammalian and drosophila citric acid cycle enzymes via iron-responsive elements.</a> Proc. Natl. Acad. Sci. USA. 93, 4925-4930 (1996).</li><li>McKie, A. T., 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+novel+duodenal+iron-regulated+transporter+IREG1,+implicated+in+the+basolateral+transfer+of+iron+to+the+circulation.">A novel duodenal iron-regulated transporter IREG1, implicated in the basolateral transfer of iron to the circulation.</a> Mol. Cell. 5, 299-309 (2000).</li><li>Gunshin, H., 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=Cloning+and+characterization+of+a+mammalian+protein-coupled+metal-ion+transporter.">Cloning and characterization of a mammalian protein-coupled metal-ion transporter.</a> Nature. 388, 482-488 (1997).</li><li>Sanchez, M., Galy, B., Muckenthaler, M. U., Hentze, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron-regulatory+proteins+limit+hypoxia-inducible+factor-2alpha+expression+in+iron+deficiency.">Iron-regulatory proteins limit hypoxia-inducible factor-2alpha expression in iron deficiency.</a> Nat. Struct. Mol. Biol. 14, 420-426 (2007).</li><li>Sanchez, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+regulation+and+the+cell+cycle:+Identification+of+an+iron-responsive+element+in+the+3'-untranslated+region+of+human+cell+division+cycle+14A+mRNA+by+a+refined+microarray-based+screening+strategy.">Iron regulation and the cell cycle: Identification of an iron-responsive element in the 3'-untranslated region of human cell division cycle 14A mRNA by a refined microarray-based screening strategy.</a> J. Biol. Chem. 281, 22865-22874 (2006).</li><li>Santos, C. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+iron+responsive+element-like+stem-loop+regulates+alpha-hemoglobin-stabilizing+protein+mRNA.">An iron responsive element-like stem-loop regulates alpha-hemoglobin-stabilizing protein mRNA.</a> J Biol Chem. 283, 26956-26964 (2008).</li><li>Liu, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Siderophore-mediated+iron+trafficking+in+humans+is+regulated+by+iron.">Siderophore-mediated iron trafficking in humans is regulated by iron.</a> J Mol Med (Berl. 90, 1209-1221 (2012).</li><li>Gray, N. 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=Recombinant+iron+regulatory+factor+functions+as+an+iron-responsive+element-binding+protein,+a+translational+repressor+and+an+aconitase.+A+functional+assay+for+translational+repression+and+direct+demonstration+of+the+iron+switch.">Recombinant iron regulatory factor functions as an iron-responsive element-binding protein, a translational repressor and an aconitase. A functional assay for translational repression and direct demonstration of the iron switch.</a> Eur. J. Biochem. 218, 657-667 (1993).</li><li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Anal. Biochem. 72, 248-254 (1976).</li><li>Testa, U., 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=Differential+regulation+of+iron+regulatory+element-binding+protein(s)+in+cell+extracts+of+activated+lymphocytes+versus+monocytes-macrophages.">Differential regulation of iron regulatory element-binding protein(s) in cell extracts of activated lymphocytes versus monocytes-macrophages.</a> J. Biol. Chem. 266, 13925-13930 (1991).</li><li>Hentze, M. W., Rouault, T. A., Harford, J. B., Klausner, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidation-reduction+and+the+molecular+mechanism+of+a+regulated+RNA-protein+interaction.">Oxidation-reduction and the molecular mechanism of a regulated RNA-protein interaction.</a> Science. 244, 357-359 (1989).</li><li>Meyron-Holtz, E. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+ablations+of+iron+regulatory+proteins+1+and+2+reveal+why+iron+regulatory+protein+2+dominates+iron+homeostasis.">Genetic ablations of iron regulatory proteins 1 and 2 reveal why iron regulatory protein 2 dominates iron homeostasis.</a> EMBO J. 23, 386-395 (2004).</li><li>Huang, F. W., Pinkus, J. L., Pinkus, G. S., Fleming, M. D., Andrews, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+model+of+juvenile+hemochromatosis.">A mouse model of juvenile hemochromatosis.</a> J. Clin. Invest. 115, 2187-2191 (2005).</li><li>Sebastiani, G., Pantopoulos, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disorders+associated+with+systemic+or+local+iron+overload:+from+pathophysiology+to+clinical+practice.">Disorders associated with systemic or local iron overload: from pathophysiology to clinical practice.</a> Metallomics. 3, 971-986 (2011).</li><li>Galy, B., Ferring, D., Hentze, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+conditional+alleles+of+the+murine+iron+regulatory+protein+(IRP)-1+and+-2+genes.">Generation of conditional alleles of the murine iron regulatory protein (IRP)-1 and -2 genes.</a> Genesis. 43, 181-188 (2005).</li><li>Gkouvatsos, 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=Iron-dependent+regulation+of+hepcidin+in+Hjv-/-+mice:+Evidence+that+hemojuvelin+is+dispensable+for+sensing+body+iron+levels.">Iron-dependent regulation of hepcidin in Hjv-/- mice: Evidence that hemojuvelin is dispensable for sensing body iron levels.</a> PLoS ONE. 9, 85530 (2014).</li><li>Goforth, J. B., Anderson, S. A., Nizzi, C. P., Eisenstein, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+determinants+within+iron-responsive+elements+dictate+iron+regulatory+protein+binding+and+regulatory+hierarchy.">Multiple determinants within iron-responsive elements dictate iron regulatory protein binding and regulatory hierarchy.</a> RNA. 16, 154-169 (2010).</li><li>Gopinath, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+of+RNA-protein+interactions.">Mapping of RNA-protein interactions.</a> Anal Chim Acta. 636, 117-128 (2009).</li></ol>

The authors declare that they have no competing financial interests.

Herein, we describe a protocol that has been developed to study the IRE-binding activities of IRP1 and IRP2, and we show representative data. By using different probes, this protocol can also be adjusted for the study of other RNA-binding proteins. A critical step is the size of the probe. Usage of long probes, which is common when the exact binding site is unknown, can result in RNA/protein complexes that do not migrate differently than the free RNA. In this case, it is advisable to remove unbound RNA by treatment with ...

A EMSA foi originalmente desenvolvido para o estudo da associação de proteínas de ligação de DNA com sequências de ADN alvo 1,2. O princípio é semelhante para as interacções de ARN / proteína 3, o que é o foco deste artigo. Resumidamente, o ARN é negativamente carregado e irá migrar em direcção ao ânodo durante a electroforese não desnaturante em gel de poliacrilamida (ou géis de agarose). A migração dentro do gel depende do tamanho do ARN, que é proporcional à sua carga. De ligação específica de uma proteína de ARN altera a sua mobilidade, e as complexo migra mais lenta em comparação com o ARN livre. Isto é devido principalmente a um aumento na massa molecular, mas também às alterações na carga e, eventualmente, conformação. Utilizando um RNA rotulado como sonda permite fácil monitoramento do &quot;atraso gel&quot; ou &quot;bandshift&quot;. Uso de 32 sondas de ARN marcadas com P é muito comum e oferece uma elevada sensibilidade. A migração de RNA / complexos de proteínas e RNA livre são detectadospor autorradiografia. Inconvenientes são o meia-vida curta de 32 P (14,29 dias), a deterioração gradual da qualidade da sonda devido a radiólise, a exigência de uma licença de radioatividade e infra-estrutura para o trabalho de radioatividade, e potenciais preocupações de biossegurança. Portanto, foram desenvolvidos métodos alternativos não isotópicas para etiquetar a sonda de RNA, por exemplo, com fluoróforos ou biotina, que permitem a detecção por imagem fluorescente ou quimioluminescente 4,5. As limitações destes métodos são o custo mais elevado e frequentemente sensibilidade reduzida em comparação com a marcação isotópica, e o potencial de marcadores não isotópicos para interferir com a interacção de ARN / proteína. Géis de poliacrilamida não desnaturantes são adequados para a maioria das aplicações de EMSA e são normalmente utilizados. Na ocasião, géis de agarose pode representar uma alternativa para a análise de grandes complexos. A principal vantagem de EMSA é que ele combina simplicidade, sensibilidade e robustez 4 &lt;/ Sup&gt;. O ensaio pode ser concluída dentro de algumas horas e não requer instrumentação sofisticada. Interacções de ARN / proteína pode ser detectada por EMSA em concentrações tão baixas como 0,1 nM ou menos, e dentro de uma ampla gama de condições de ligação (pH 4,0-9,5, a concentração de sal monovalente 1-300 mM, e a temperatura 0-60 ° C). ARN / proteína a formação do complexo também pode ser estudada pelo ensaio de ligação ao filtro. Este é um procedimento simples, rápido e barato, baseado na retenção dos complexos de ARN / proteína em um filtro de nitrocelulose, enquanto que uma sonda de RNA livre passa por 6. Em comparação com a EMSA, é limitado nos casos em que a sonda de ARN contém múltiplos locais de ligação, ou o extracto em bruto contém mais do que uma proteína de ligação a ARN que se ligam a sonda no mesmo local. Enquanto múltiplas interacções ARN / proteína vai escapar à detecção por ensaio de ligação ao filtro, que pode ser facilmente visualizado por EMSA. Em alguns casos, a visualização é vésperan possível quando dois complexos de ARN / proteína co-migrar (por exemplo, humano IRP1 / IRE e IRP2 / complexos IRE), por adição de um anticorpo contra uma das proteínas de ligação a ARN para a reacção de EMSA, obtendo-se ainda mais sobre o gel de retardo ( &quot;supershift&quot;) 7. O EMSA tem sido amplamente utilizado para estudar e IRP1 IRP2, que são reguladores pós-transcricional do metabolismo do ferro 8-10. Eles operam através da ligação a IRES, estruturas hairpin filogeneticamente conservados dentro das UTRs de vários mRNAs 11. IRES foram descobertos pela primeira vez nos mRNAs que codificam 12 ferritina e transferrina receptor 1 (TfR1) 13, proteínas de armazenamento de ferro e absorção, respectivamente. Mais tarde, IRES foram encontrados em específico-erythroid aminolevulinato sintase (ALAS2) 14, aconitase mitocondrial 15, ferroportin 16, transportador de metal divalente 1 (DMT1) 17, hipóxia fator induzível 2 <em&gt; α (HIF2α) 18, e outro mRNAs 19-21. O protótipo H- e mRNAs L-ferritina conter um IRE na sua UTR 5 &#39;, enquanto TfR1 ARNm contém múltiplos IRES em sua extremidade 3&#39; UTR. Interações IRE / PIA especificamente inibir a tradução do mRNA ferritina por estericamente bloqueando a sua associação dos 43S da subunidade ribossômica; Além disso, eles estabilizar TfR1 mRNA contra clivagem endonucleolítica. IRP1 e share IRP2 extensa seqüência de similaridade e apresentam alta atividade de ligação IRE em células sedentos de ferro. Em células repletas de ferro, IRP1 monta um aglomerado cubano Fe-S que converte para aconitase citosólico em detrimento da sua actividade de ligação ao IRE, enquanto IRP2 sofre degradação proteossoma. Assim, as interacções IRE / PIA depender do estado celular de ferro, mas também são regulados por outros sinais, tais como H 2 O 2, o óxido nítrico (NO) ou hipoxia. Aqui, descrevemos o protocolo para avaliar a atividade IRE de ligação a partir de extratos de células de tecido cru e por eMSA. Utilizou-se um marcado com 32 P H-ferritina sonda IRE que foi gerado por transcrição in vitro a partir de um molde de ADN de plasmídeo (I-12.CAT), onde a sequência IRE foi originalmente introduzido na orientação com sentido a jusante do local da polimerase de ARN de T7 pela clonagem de oligonucleótidos sintéticos 22 recozidos.

<table border="0" cellpadding="0" cellspacing="0" width="430">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:195px;">Name of the Materials&#160;</td>
			<td style="width:119px;">Company</td>
			<td style="width:117px;">Catalog Number&#160;</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">leupeptin</td>
			<td>SIGMA</td>
			<td>L2884</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">PMSF</td>
			<td>SIGMA</td>
			<td align="right">78830</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">BioRad Protein Assay</td>
			<td>BIORAD</td>
			<td>500-0006</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">T7 RNA polymerase</td>
			<td>Thermoscientific</td>
			<td>EPO111</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Rnase Inhibitor</td>
			<td>Invitrogen</td>
			<td>15518-012</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">UTP [alpha-32P]</td>
			<td>Perkin-Elmer</td>
			<td>NEG507H</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Scintillation liquid</td>
			<td>Beckman Coulter</td>
			<td align="right">141349</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">heparin</td>
			<td>SIGMA</td>
			<td>H0777</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Rnase T1</td>
			<td>Thermoscientific</td>
			<td>EN0541</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Name of the Equipments&#160;</td>
			<td>Company</td>
			<td>Catalog Number&#160;</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Tissue Ruptor</td>
			<td>Qiagen</td>
			<td align="right">9001271</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Scintillation counter</td>
			<td>Beckman Coulter</td>
			<td>LS6500</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Protean II xi Cell&#160;&#160;</td>
			<td>BIORAD</td>
			<td>165-1834</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">20 wells combs 1.5mm thick</td>
			<td>BIORAD</td>
			<td>165-1868</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">1.5 mm spacers</td>
			<td>BIORAD</td>
			<td>165-1849</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">PowerPac</td>
			<td>BIORAD</td>
			<td>164-5070</td>
		</tr>
	</tbody>
</table>

electrophoretic mobility shift assay (emsa) for the study of rna-protein interactions: the ire/irp example

Interações RNA / proteínas são fundamentais para vias de regulação pós-transcricional. Entre as proteínas de ligação a ARN citosólicos melhor caracterizados são proteínas reguladoras de ferro, e IRP1 IRP2. Eles ligam-se a elementos responsivos de ferro (IRES) dentro das regiões não traduzidas (UTRs) de vários mRNAs alvo, controlando assim a tradução de mRNAs ou estabilidade. Interações IRE / PIA têm sido amplamente estudados pela EMSA. Aqui, nós descrevemos o protocolo de EMSA para analisar a actividade de ligação de IRE-IRP1 e IRP2, que pode ser generalizado para avaliar a actividade de outras proteínas de ligação a ARN, bem. Um lisato de proteína bruto contendo uma proteína de ligação de ARN, ou uma preparação purificada desta proteína, é incubado com um excesso de 32 sonda de ARN marcada com P, para permitir a formação do complexo. A heparina é adicionado para impedir a proteína não específica para sondar ligação. Subsequentemente, a mistura é analisada por electroforese não desnaturante em gel de poliacrilamida. A sonda livremigra rapidamente, enquanto que o ARN / proteína apresenta complexos de mobilidade retardada; daí, o procedimento também é chamado de &quot;retardamento em gel&quot; ou ensaio &quot;bandshift&quot;. Depois de completada a electroforese, o gel é seco e complexos de ARN / proteína, bem como sonda livre, são detectados por auto-radiografia. O objetivo geral do protocolo é para detectar e quantificar IRE / IRP e outras interações RNA / proteína. Além disso, a EMSA também pode ser utilizado para determinar a especificidade, a afinidade de ligação, e a estequiometria da interacção de ARN / proteína sob investigação.

A radiolabeled IRE probe was prepared, as described in sections 3 and 4 of the protocol. The sequence of the probe was 5&#39;-GGGCGAAUUC GAGCUCGGUA CCCGGGGAUC CUGCUUCAAC AGUGCUUGGA CGGAUCCU-3&#39;; the bolded nucleotides represent an unpaired C residue and the loop, which are critical IRE features. The specific radioactivity of the probe was 4.5 x 109 cpm/&#956;g of RNA.
To assess the effects of iron perturbations on IRE-binding activity, murine RAW...

Watch this Scientific Journal Video about Electrophoretic Mobility Shift Assay EMSA for the Study of RNA-Protein Interactions: The IRE/IRP Example at JoVE.com

Electrophoretic Mobility Shift Assay EMSA for the Study of RNA-Protein Interactions: The IRE/IRP Example

Here we present a protocol to analyze RNA/protein interactions. The electrophoretic mobility shift assay (EMSA) is based on the differential migration of RNA/protein complexes and free RNA during native gel electrophoresis. By using a radiolabeled RNA probe, RNA/protein complexes can be visualized by autoradiography.

Electrophoretic Mobility Turno Assay (EMSA) para o estudo das interações RNA em proteínas: O IRE / IRP Exemplo

RNA/protein interactions are critical for post-transcriptional regulatory pathways. Among the best-characterized cytosolic RNA-binding proteins are iron ...

electrophoretic-mobility-shift-assay-emsa-for-study-rna-protein

Research

JoVE Journal

Biology

Electrophoretic Mobility Shift Assay (EMSA) for the Study of RNA-Protein Interactions: The IRE/IRP Example

53.2K visualizações.  Jewish General Hospital. Here we present a protocol to analyze RNA/protein interactions. The electrophoretic mobility shift assay (EMSA) is based on the differential migration of RNA/protein complexes and free RNA during native gel electrophoresis. By using a radiolabeled RNA probe, RNA/protein complexes can be visualized by autoradiography.

Vídeo: Electrophoretic Mobility Shift Assay EMSA for the Study of RNA-Protein Interactions: The IRE/IRP Example

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Teste de sensibilidade visual para a velocidade e direção do movimento em Lagartos

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Biologia

Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, branching, lumen formation, anastomosis, and formation of a new basement membrane.  Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels within the assays often do not resemble vessels in vivo.  Based on earlier work by Nehls and Drenckhahn, we have optimized an in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts.  This model recapitulates all of the key early stages of angiogenesis and, importantly, the vessels display patent intercellular lumens surrounded by polarized EC. EC are coated onto cytodex microcarriers and embedded into a fibrin gel.  Fibroblasts are layered on top of the gel where they provide necessary soluble factors that promote EC sprouting from the surface of the beads.  After several days, numerous vessels are present that can easily be observed under phase-contrast and time-lapse microscopy. This video demonstrates the key steps in setting up these cultures.

This video demonstrates the protocol of an in vitro angiogenesis assay that recapitulates several stages of angiogenesis. Time-lapse images of sprouting, lumen formation, branching and anastomosis - key features of angiogenesis - are shown.

Otimizado de fibrina Gel Assay Bead para o Estudo da angiogênese

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These ...

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

Gerados por computador Modelo Estímulos animal

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

Do-It-Yourself Dispositivo de Recuperação de amostras criopreservadas cair acidentalmente em Tanques de armazenamento criogênico

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

A fase líquida ensaio de captura de afinidade usando pérolas magnéticas para estudar interação proteína-proteína: O Exemplo Poliovírus Nanobody-

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

A Plataforma de Produção de Proteína Complexo MultiBac no EMBL

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

Brotando Yeast extração e purificação de proteínas para o estudo da função, interações e modificações pós-traducionais

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

Combinando uma única molécula de Manipulação e imagem para o estudo das interações proteína-DNA

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

Ensaio para imunoprecipitação da cromatina a identificação de Arabidopsis interações proteína-ADN<em&gt; In Vivo</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay (EMSA) and DNA-affinity Precipitation Assay (DAPA)

Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical disease or phenotype. For many diseases and traits, most variants are non-coding, and are thus likely to act by impacting subtle, comparatively hard to predict mechanisms controlling gene expression. Here, we describe a general strategic approach to prioritize non-coding variants, and screen them for their function. This approach involves computational prioritization using functional genomic databases followed by experimental analysis of differential binding of transcription factors (TFs) to risk and non-risk alleles. For both electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genetic variants, a synthetic DNA oligonucleotide (oligo) is used to identify factors in the nuclear lysate of disease or phenotype-relevant cells. For EMSA, the oligonucleotides with or without bound nuclear factors (often TFs) are analyzed by non-denaturing electrophoresis on a tris-borate-EDTA (TBE) polyacrylamide gel. For DAPA, the oligonucleotides are bound to a magnetic column and the nuclear factors that specifically bind the DNA sequence are eluted and analyzed through mass spectrometry or with a reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blot analysis. This general approach can be widely used to study the function of non-coding genetic variants associated with any disease, trait, or phenotype.

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay EMSA and DNA-affinity Precipitation Assay DAPA

We present a strategic plan and protocol for identifying non-coding genetic variants affecting transcription factor (TF) DNA binding. A detailed experimental protocol is provided for electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genotype-dependent TF DNA binding.

Triagem para Funcional não-codificante variantes genéticas Usando eletroforética Mobilidade Mudança Assay (EMSA) e DNA afinidade Precipitation Assay (DAPA)

Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical ...