Biz değerli tavsiyeler için Victor GJ Rodgers için çok müteşekkiriz. Biz çok yakın ortak çalışma ve bu çalışma ile yardım için Liao grubun tüm üyeleri teşekkür ederiz. Bu çalışma, Ulusal Sağlık Enstitüleri (JL Grant AI076504) tarafından desteklenmiştir.

1. Plazmid Oluşumlar <ol><li> PCR ile genlerin açık okuma çerçeveleri Kuvvetlendirme ve PCRII TOPO-vektörüne klonlanması PCR ürünlerinin. </li><li> Dizilenerek teyit ürünleri, ve klon kodlayan cDNA CyPet-(pre-SUMO1)-YPet, CyPet-SUMO1, YPet ve bir N-terminal hexahistidine etiket ile pET28 (b) vektörü içine SENP1 katalitik etki. </li></ol> 2. Protein Ekspresyonu ve Saflaştırılması <ol><li> PET28 ile suşu BL21 (DE3) ve Escherichia coli hücreleri Transform (b) CyPet-(pre-SUMO1)-YPet, CyPet-SUMO1, YPet ve SENP1 bir katalitik etki kodlayan vektörler. </li><li> 600 nm&#39;de 0.5 bir optik yoğunluğa ulaşmak üzere 37 ° C (250 rpm&#39;de sallama), 3 saat boyunca 2xYT ortamı içinde yeniden izlenmesiyle dönüştürülmüş bakteriler yetiştirilir. </li><li> Izopropil-β-D-thiogalactoside (IPTG) (nihai konsantrasyon) proteini ifade etme indüklemek ve 25 saat için sallayın 16 ° C ile 200 rpm ile 100 uM ekleyin. </li><li> Centrifug bakterilerin hasat4 10.000 rpm&#39;de 5 dk tirme ° ve 20 mM Tris-HCl (pH 7.4), 50 mM NaCl, 5 mM imidazol ve bir tampon içinde tekrar süspansiyon için onları C. </li><li> MiSonics 4.000 sonikatör kullanılarak 25 W bir güç ayarında 5 saniye aralıklarla 10 dakika için hücreler sonikasyon ve 4 35,000 x g&#39;de santrifüj ile bunları toplamaya ° C&#39;de 30 dakika süre ile. </li><li> 1 L kültür için 500 ml Ni-NTA boncuklar ile sütun ayarlama ve sütuna süpernatant aktarmak. </li><li> 20 mM Tris-HCl (pH 7.4), 500 mM NaCl, ve iki defa 10 mM imidazol tampon ile reçine yıkanır. </li><li> 20 mM Tris-HCl (pH 7.4), 500 mM NaCl, 500 mM imidazol ile ve 20 mM Tris-HCl (pH 7.4), 50 mM NaCl ve 1 mM ditiotreitol (DTT) de bir tampon içine diyaliz tamponu ile elute protein 4 ° C gecede. </li><li> Bradford tahlili ile saflaştırılmış protein konsantrasyonu belirlenir. Protein konsantrasyonu ölçümleri için alternatif yöntem SDS-PAGE jel elektroforezi olmak ve sonra Coomassie mavisi ile boyandı olacak görüntüleme ile takipniceliksel. </li></ol> 3. Kantitatif Spektrum Analizi FRET <ol><li> Tahlil için genel strateji (Şekil 1) sinyalizasyon Fret dayanmaktadır. FRET çifti, CyPet ve YPet, ön-SUMO1, sırasıyla N-ve C-termini ile etiketlendi. SENP1 keser füzyon SUMO1 en C-terminusunda Gly-Gly yerinde protein CyPet-(pre-SUMO1)-YPet ve, böylece, YPet ile SUMO kuyruk serbest bırakır. Sinyal CyPet gelen emisyon artış ve CyPet dalgaboyu az YPet emisyon dramatik bir azalma ile sonuçlanan, bozulur FRET. </li><li> Mutlak FRET indüklenen YPet emisyon, CyPet doğrudan emisyon ve YPet doğrudan emisyon (Şekil: 530 nm dalga boyunda ışık ile 414 nm, toplam floresans emisyon CyPet-(pre-SUMO1)-YPet heyecanlı zaman üç kaynaktan elde edilebilir 2). </li></ol><img alt="Denklem 1" src="/files/ftp_upload/4430/4430eq1.jpg" /> nerede FL 530/414 &lt;/ Sub&gt; 414 nm&#39;de uyarıldığı 530 nm&#39;de toplam floresans emisyonu, FL FRET mutlak sinyal FRET olduğunu, FL CyPet (devam) 414 nm&#39;de heyecanlı CyPet doğrudan emisyon ve FL YPet (devam) YPet olduğunu 414 nm&#39;de uyarıldığı zaman emisyon yönlendirebilirsiniz. Ve simge (devam) katkı anlamına gelir. <ol start="3"><li> Α sabit bir oran ile 414 nm&#39;de uyarıldığı zaman 530 nm CyPet arasında doğrudan emisyonu 475 nm&#39;de emisyon orantılı idi. CyPet-SUMO1 50 konsantrasyonları, 100, 200, 500, ve 750 nM hazırlanan ve 1 mM edildi ve 475 ve 530 nm emisyon α (Şekil 3-1) belirlemek için 414 nm&#39;de uyarılma sonrasında ölçüldü. </li><li> Β sabit bir oran ile 475 nm&#39;de uyarıldığı zaman 414 nm uyarma altında 530 nm&#39;de YPet arasında doğrudan emisyonu 530 nm&#39;de emisyon orantılı idi. YPet 50 konsantrasyonları, 100, 200, 500, 750 hazırlandıörnekleri β (Şekil 3-2) belirlemek için 414 dalga boylarında ve 475 nm ile heyecanlı iken nd 1.000 nM ve 530 nm&#39;de emisyon ölçüldü. </li><li> CyPet-(pre-SUMO1)-YPet SENP1 tarafından sindirilir zaman, dilinim CyPet-SUMO1 ve YPet ile SUMO1 kuyruk yayımladı. Bileşik 414 nm&#39;de uyarıldığı zaman, 530 nm (FL &#39;530/414) de floresans emisyonu azalmış ama hala gibi üç bölüme ayrılabilir: </li></ol><img alt="Denklem 2" src="/files/ftp_upload/4430/4430eq2.jpg" /> FL &#39;414 nm&#39;de uyarıldığı zaman 530/414 sindirim sonra 530 nm&#39;de toplam floresans emisyonu, FL&#39; burada kalan mutlak FRET sinyal FRET, FL &#39;CyPet (475/414) iken sindirim sonra 475 nm&#39;de CyPet emisyonu 414 nm (burada CyPet emisyon iki bölümden alınmıştır: CyPet-(pre-SUMO1)-YPet sindirilmemiş ve CyPet-SUMO1 sindirilmiş) heyecanlı ve FL YPet(530/475) CyPet-(pre-SUMO1)-YPet sindirilmiş olup olmadığını sabittir 475 nm&#39;de heyecanlı YPet emisyon vardır. <ol start="6"><li> SENP1 ile sindirim sonra, geri kalan emisyon (FL &#39;FRET)&#39; dir FRET: </li></ol><img alt="Denklem 3" src="/files/ftp_upload/4430/4430eq3.jpg" /> C CyPet-(pre-SUMO1)-YPet ve x &#39;in toplam konsantrasyonunu sindirilmiş konsantrasyonu CyPet-(pre-SUMO1)-YPet olup. <ol start="7"><li> Tüm öğeleri birleştirerek, 414 nm (FL &#39;530/414) ve uyarma altında 530 nm&#39;de tespit floresans emisyonu: </li></ol><img alt="Denklem 4" src="/files/ftp_upload/4430/4430eq4.jpg" /> 4. Enzim Kinetik Çalışması için FRET tabanlı Proteaz Assay <ol><li> CyPet-(pre-SUMO1)-YPet (20 mM Tris-HCl (pH 7.4), 50 mM NaCl,% 0.1 bir tampon içinde 37 ° C &#39;de SENP1 arasında katalitik alan ile inkübe edildi v /v) Tween-20, 80 ml toplam hacim 1 mM DTT ve 384-plaka içine aktarılır. </li><li> Runs 15 sn aralıklarla 5 dakika süreyle bir floresan multiwell plaka okuyucu içinde 414 nm de bir uyarım sonrası 475 ve 530 nm&#39;de floresans emisyon ölçümü ile yapılmıştır. </li><li> Reaksiyon hızı (v) olarak alt tabaka (S) miktarı değişim ile korelasyon: </li></ol><img alt="Denklem 5" src="/files/ftp_upload/4430/4430eq5.jpg" /><ol start="4"><li> Ürünün konsantrasyonu olarak 0 dan üstel olarak artmaktadır [S] 0 (orijinal substrat konsantrasyonu), t = 0: </li></ol><img alt="Denklem 6" src="/files/ftp_upload/4430/4430eq6.jpg" /><ol start="5"><li> T = 0, orijinal hızı (V 0) olduğunda: </li></ol><img alt="Denklem 7" src="/files/ftp_upload/4430/4430eq7.jpg" /><ol start="6"><li> KonsSENP1 arasında entration tespit edildi, ve CyPet-(pre-SUMO1)-YPet konsantrasyonu Michaelis-Menten denklem için gerekli olan enzim konsantrasyonu, 100 kat daha fazla çıkarılmıştır. </li><li> Floresan okumaların tüm kantitatif FRET analizi yöntemi ile analiz ve Michaelis-Menten eşitliği sığdırmak için GraphPad Prism V Yazılım aktarılmıştır. Doğrusal olmayan regresyon zamanda tablolama programları (Microsoft Excel gibi) gibi daha yaygın yazılım paketleri yardımı ile yapılabilir. </li></ol> 5. Temsilcisi Sonuçlar SENP1 ön-SUMO1 olgunlaşması süreci sırasında 475 ve 530 nm de floresan sinyalini meydana gelen değişiklikleri takip edilerek tespit edilebilir. Sonuçlar, bir alt tabaka doza bağlı olarak SENP1 tarafından önceden SUMO1 sindirim hızı (Şekil 4). Bu da gösteriyor öncesi SUMO1 en olgunlaşması için SENP1 ve katalitik etki sergiler mükemmel aktivite olduğunu. Başlangıç ​​reaksiyontion hızları farklı substrat konsantrasyonları (Tablo 1) ile ilgili olarak yukarıda analizi ile hesaplanmıştır. K kedi / K M oranı, genel olarak bir alt tabaka veya başka substratlar ile, belirli bir enzim ile farklı enzim etkinliği karşılaştırmak için kullanılır. K M ve V max karşılık gelen çeşitli ilk hızlar, çizilerek Michaelis-Menten denklemden elde edilebilir . CyPet-(pre-SUMO1)-YPet (Şekil 5) k kedi halinde elde edildi farklı konsantrasyonları için: <img alt="Denklem 8" src="/files/ftp_upload/4430/4430eq8.jpg" /> Yukarıdaki analize göre, hesaplanan K M 0.21 idi ± 0.04 uM, k kedi ± 0.28 s -1 6.90 idi ve k cat / K M oranı (± 0.55 3.2) idi x10 7 M -1 </sup<html> &gt; S -1. <img alt="Şekil 1" src="/files/ftp_upload/4430/4430fig1.jpg" /> Şekil 1. SENP öncesi sumos olgunlaşması için FRET tabanlı proteaz tahlil grafiği. <img alt="Şekil 2," src="/files/ftp_upload/4430/4430fig2.jpg" /> Şekil 2. Donör, alıcı ve FRET tarafından konulan sermaye olarak floresan sinyal kantitatif analizi. CyPet-414 nm uyarma altında (pre-SUMO1)-YPet. FL CyPet (530/414) gelen emisyon spektrumları diseksiyonu 414 nm uyarma altında 530 nm&#39;de CyPet emisyon, FL FRET olduğu FRET indüklenen YPet 414 nm uyarma altında 530 nm&#39;de emisyon ve FL YPet (530/414) 414 nm uyarma altında 530 nm&#39;de YPet emisyon olduğunu. load/4430/4430fig3.jpg &quot;/&gt; Şekil 3. Yön emisyon faktörü α ve β hesaplanması. <img alt="Şekil 4," src="/files/ftp_upload/4430/4430fig4.jpg" /> Şekil 4. SENP1 ve katalitik etki farklı oranlarda tarafından sindirilir CyPet-(pre-SUMO1)-YPet kantitatif analizi. Reaksiyonlar ilk 5 dakika içerisinde izlenmiştir. <img alt="Şekil 5," src="/files/ftp_upload/4430/4430fig5.jpg" /> Şekil 5. SENP1 tarafından CyPet-(pre-SUMO1)-YPet en sindirim Michaelis-Menten grafik analizi. Veri çizilen ve GraphPad Prism V ve doğrusal olmayan regresyon ile analiz edildi. <table border="1"><tbody><tr><td> [S] (uM) </td><td> V 0 (uM / s) </td></tr><tr><td> 0.115 </td><td> 0.0023 ± 0.00005 </td></tr&gt; <tr><td> 0.214 </td><td> 0,0028 ± 0,00004 </td></tr><tr><td> 0.407 </td><td> 0.0033 ± 0.00007 </td></tr><tr><td> 0.594 </td><td> 0.0037 ± 0,00013 </td></tr><tr><td> 0.725 </td><td> 0.0043 ± 0.00012 </td></tr><tr><td> 1.471 </td><td> 0,0051 ± 0,00036 </td></tr><tr><td> 1.899 </td><td> 0.0050 ± 0.00031 </td></tr><tr><td> 2.300 </td><td> 0.0050 ± 0,00062 </td></tr></tbody></table> Tablo 1. SENP1 öncesi SUMO1 olgunlaşan ilk hızlarının belirlenmesi. Her bir alt tabaka konsantrasyonu, dört numune sindirim ölçmek için kullanılmıştır. Standart sapma, bu dört örneklerin varyasyonları geldi. <table border="1"><tbody><tr><td> K M (uM) </td><td> k cat (s-1) </td><td> k cat / k M (M -1 • s-1) </td></tr><tr><td> 0.21 ± 0.04 </td><td> 6.90 ± 0.28 </td><td> 3.2 ± 0.55 × 10 7 </td></tr></tbody></table> Tablo 2. Kantitatif tarafından SENP1 öncesi SUMO1 olgunlaşan Kinetik parametrelerin analiz FRET. Standart sapma, her bir alt tabaka konsantrasyonu olarak dört numune geldi.

<ol>
	<li>Johnson, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+modification+By+SUMO.">Protein modification By SUMO.</a> Annual Review of Biochemistry. 73, 355-382 (2004).</li><li>Gill, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SUMO+and+ubiquitin+in+the+nucleus:+different+functions,+similar+mechanisms.">SUMO and ubiquitin in the nucleus: different functions, similar mechanisms.</a> Genes &amp; Development. 18, 2046-2059 (2004).</li><li>Hay, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SUMO:+a+history+of+modification.">SUMO: a history of modification.</a> Molecular Cell. 18, 1-12 (2005).</li><li>M&#252;ller, S., Hoege, C., Pyrowolakis, G., Jentsch, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SUMO,+ubiquitin's+mysterious+cousin.">SUMO, ubiquitin's mysterious cousin.</a> Nature. , 202-210 (2001).</li><li>Hochstrasser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SP-RING+for+SUMO:+new+functions+bloom+for+a+ubiquitin-like+protein.">SP-RING for SUMO: new functions bloom for a ubiquitin-like protein.</a> Cell. , 5-8 (2001).</li><li>Drag, M., Salvesen, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DeSUMOylating+enzymes-SENPs.">DeSUMOylating enzymes-SENPs.</a> IUBMB Life. 60, 734-742 (2008).</li><li>Mukhopadhyay, D., Dasso, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modification+in+reverse:+the+SUMO+proteases.">Modification in reverse: the SUMO proteases.</a> Trends in Biochemical Sciences. 32, 286-295 (2007).</li><li>Reverter, D., Lima, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+for+SENP2+protease+interactions+with+SUMO+precursors+and+conjugated+substrates.">Structural basis for SENP2 protease interactions with SUMO precursors and conjugated substrates.</a> Nature Structural &amp; Molecular Biology. 13, 1060-1068 (2006).</li><li>Shen, 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=SUMO+protease+SENP1+induces+isomerization+of+the+scissile+peptide+bond.">SUMO protease SENP1 induces isomerization of the scissile peptide bond.</a> Nature Structural &amp; Molecular Biology. 13, 1069-1077 (2006).</li><li>Horton, R., Strachan, E., Vogel, K., Riddle, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+substrate+for+deubiquitinating+enzymes+based+on+time-resolved+fluorescence+resonance+energy+transfer+between+terbium+and+yellow+fluorescent+protein.">A substrate for deubiquitinating enzymes based on time-resolved fluorescence resonance energy transfer between terbium and yellow fluorescent protein.</a> Analytical Biochemistry. 360, 138-143 (2007).</li><li>Mikolajczyk, 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=Small+Ubiquitin-related+Modifier+(SUMO)-specific+Proteases:+PROFILING+THE+SPECIFICITIES+AND+ACTIVITIES+OF+HUMAN+SENPs.">Small Ubiquitin-related Modifier (SUMO)-specific Proteases: PROFILING THE SPECIFICITIES AND ACTIVITIES OF HUMAN SENPs.</a> Journal of Biological Chemistry. 282, 26217-26224 (2007).</li><li>Kolli, N., Mikolajczyk, J., Drag, M., Mukhopadhyay, D., Moffatt, N., Dasso, M., Salvesen, G., Wilkinson, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distrubition+and+paralogue+specificity+of+mammalian+deSUMOylating+enzymes.">Distrubition and paralogue specificity of mammalian deSUMOylating enzymes.</a> Biochem. J. , 335-344 (2010).</li><li>Drag, M., Mikolajczyk, J., Krishnakumar, I. M., Huang, Z., Salvesen, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity+profiling+of+human+deSUMOylating+enzymes+(SENPs)+with+synthetic+substrates+suggests+an+unexpected+specificity+of+two+newly+characterized+members+of+the+family.">Activity profiling of human deSUMOylating enzymes (SENPs) with synthetic substrates suggests an unexpected specificity of two newly characterized members of the family.</a> Biochemical Journal. 409, 461 (2008).</li><li>Engels, I. 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=A+time-resolved+fluorescence+resonance+energy+transfer-based+assay+for+DEN1+peptidase+activity.">A time-resolved fluorescence resonance energy transfer-based assay for DEN1 peptidase activity.</a> Analytical Biochemistry. 390, 85-87 (2009).</li><li>Martin, S., Hattersley, N., Samuel, I., Hay, R., Tatham, 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+fluorescence-resonance-energy-transfer-based+protease+activity+assay+and+its+use+to+monitor+paralog-specific+small+ubiquitin-like+modifier+processing.">A fluorescence-resonance-energy-transfer-based protease activity assay and its use to monitor paralog-specific small ubiquitin-like modifier processing.</a> Analytical Biochemistry. 363, 83-90 (2007).</li><li>Nguyen, A. W., Daugherty, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+optimization+of+fluorescent+proteins+for+intracellular+FRET.">Evolutionary optimization of fluorescent proteins for intracellular FRET.</a> Nature Biotechnology. 23, 355-360 (2005).</li></ol>

Çıkar çatışması ilan etti.

FRET teknoloji SENP1 9 tarafından önceden SUMO1 olgunlaşan incelemek için kullanılmıştır. CFP-YFP, verici ile alıcı emilim oranı olarak FRET çifti ve rasyometrik analizi kullanılmıştır kinetik özelliklerini karakterize etmek için kullanıldı. Bununla birlikte, geleneksel rasyometrik içinde kendinden flüoresan analiz FRET verici ve alıcı arasında hiç düşünülmemiştir. Oranı doğrudan sindirilmiş substrat miktarı ile korele değildir. Burada SENP1 ön...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td></tr><tr><td> PCR II TOPO Takımı </td><td> Invitrogen </td><td> K466040 </td></tr><tr><td> 2xYT </td><td> Araştırma Ürünleri Internationa.l Corp </td><td> X15600 </td></tr><tr><td> Ni-NTA Agrose </td><td> Thermo Scientific </td><td> 88222 </td></tr><tr><td> Coomassie artı (Bradford) Assay Regent </td><td> Thermo Scientific </td><td> 23238 </td></tr><tr><td> 384-plaka (cam alt) </td><td> Greiner </td><td> 781892 </td></tr><tr><td> FlexStation II 384 plaka okuyucu </td><td> Moleküler Cihaz </td><td></td></tr></tbody></table>

quantitative fret (f&#246;rster resonance energy transfer) analysis for senp1 protease kinetics determination

Ubikitin veya ubikitin-benzeri proteinler (Ubls) ile proteinlerin tersinir posttranslasyonel değişiklikler yaygın olarak dinamik bir protein aktivitesini düzenleyen ve çok biyolojik olarak farklı roller için kullanılır. Örneğin, SUMO kovalent hücre siklusunun regülasyonunda, hücre yaşam ve ölümü, DNA hasar cevabı ve stres yanıtın 1-5 gibi birçok hücresel süreçlerin önemli rolü olan bir büyük numarası veya proteinleri değiştirir. SENP, SUMO-spesifik proteaz olarak, SUMO öncüllerinin olgunlaşmasının endopeptidazla olarak veya isopeptidase gibi fonksiyonları hedef proteinleri SUMO kaldırmak ve sumoilasyon döngüsü 1,3,6,7 yenilemek için. Bir enzimin katalitik etkinliği ya da en iyi seçicilik kinetik sabitler, k kedi / K M oranı ile karakterize edilir. Çeşitli çalışmalarda, SUMO-SENP çiftleri kinetik parametreleri rad, poliakrilamid jel tabanlı western-blot dahil olmak üzere çeşitli yöntemlerle tespit edilmiştirioactive-etiketli substrat, floresan bileşik ya da protein alt tabaka 8-13 etiketli. Ancak, &quot;yerli&quot; proteinleri kullanılır ama poliakrilamid-jel tabanlı teknikler, zahmetli ve teknik olarak zor olan, kolayca ayrıntılı nicel analiz için kendilerini borç yoktur. ACC (7-amino-4-carbamoylmetylcoumarin) ya da AMC (7-amino-4-methylcoumarin) florofor ile tetrapeptides ya da proteinlerin kullanıldığı çalışmalar k kedi / K M edilen doğal substratlar göre büyüklük daha düşük en fazla iki sipariş ya idi veya açıkça SENPs ve iso-ve endopeptidazla faaliyetleri ayırt edemez. Son zamanlarda, FRET tabanlı bir proteaz deneyleri mavi floresan protein FRET çifti (CFP) ve sarı floresan protein (YFP) 9,10,14,15 ile deubiquitinating enzimler (Dubs) ya da SENPs incelemek için kullanılmıştır. Proteaz ac için sinyal monitör FRET için donör emisyonu alıcı emisyon oranı ölçülebilir bir parametre olarak kullanılmıştırbilirlik belirlenmesi. Bununla birlikte, bu yöntem alıcı ve verici kendi flüoresans ile alıcı ve verici emisyon dalga boyunda çalışan sinyal çapraz bulaşma göz ardı edilir ve böylece doğru değildir. Bu SENP1 ön-SUMO1 olgunlaşma kinetik parametrelerinin belirlenmesi için bir yeni ve son derece hassas kantitatif FRET bazlı Proteaz analiz geliştirilmiştir. Bir tasarlanmış, önemli ölçüde geliştirilmiş FRET verim ve floresan kuantum verimi ile çift CyPet ve YPet FRET CyPet-(pre-SUMO1)-YPet alt tabaka 16 oluşturmak için kullanılmıştır. Biz farklılaşmış ve verici ve alıcı tarafından katkı ve sırasıyla alıcı ve emisyon dalga boylarında FRET mutlak floresans sinyalleri sayılabilir. K kedi / K M değeri SENP1 x10 7 M -1 s -1 genel enzim kinetik parametreler ile uyum içindedir ön SUMO1, karşı (3.2 ± 0.55) olarak elde edildi. Bu nedenle, bu yöntem geçerlidir ve bir de kullanılabilirsa genel hem de diğer proteazlar karakterize yaklaşım.

Watch this Scientific Journal Video about Quantitative FRET FÃ¶rster Resonance Energy Transfer Analysis for SENP1 Protease Kinetics Determination at JoVE.com

Quantitative FRET F&amp;#246;rster Resonance Energy Transfer Analysis for SENP1 Protease Kinetics Determination

FRET kantitatif analizi (Förster rezonans enerji transferi) sinyalleri içeren bir roman yöntemini enzim kinetiği çalışmaları için tanımlanmıştır.<em&gt; K<sub&gt; M</sub</em&gt; Ve<em&gt; K<sub&gt; Kedi</sub</em&gt; (Küçük ubikitin gibi değiştirici) ön SUMO1 için SENP1 (SUMO / Sentrin belirli proteaz 1) &#39;in katalitik alanının hidroliz için elde edilmiştir. Bu kantitatif-FRET bazlı proteaz kinetik çalışma genel ilkeler diğer proteazlar için uygulanabilir.

Kantitatif SENP1 Proteaz Kinetik Belirlenmesi (Förster rezonans enerji transferi) Analizi FRET

Reversible posttranslational modifications of proteins with ubiquitin or ubiquitin-like proteins (Ubls) are widely used to dynamically regulate protein ...

quantitative-fret-frster-resonance-energy-transfer-analysis-for-senp1

Research

Education

JoVE Journal

JoVE Core

Physics

Bioengineering

Quantitative FRET (F&#246;rster Resonance Energy Transfer) Analysis for SENP1 Protease Kinetics Determination

Unit 1

Work and Kinetic Energy

SCIENTISTS IN ACTION

18.9K Görüntüleme.  University of California, Riverside. FRET kantitatif analizi (Förster rezonans enerji transferi) sinyalleri içeren bir roman yöntemini enzim kinetiği çalışmaları için tanımlanmıştır. K M

Video: Quantitative FRET F&#246;rster Resonance Energy Transfer Analysis for SENP1 Protease Kinetics Determination

Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated micro-plates. Under these conditions, cells spread and divide in all directions resulting in an inherent variability in cell shape, morphology and behavior. The high cell-to-cell variance of the overall population impedes the success of HCA, especially for drug development. The ability of micropatterns to normalize the shape and internal polarity of every individual cell provides a tremendous opportunity for solving this critical bottleneck 1-2.
To facilitate access and use of the micropatterning technology, CYTOO has developed a range of ready to use micropatterns, available in coverslip and microwell formats. In this video article, we provide detailed protocols of all the procedures from cell seeding on CYTOOchip micropatterns, drug treatment, fixation and staining to automated acquisition, automated image processing and final data analysis. With this example, we illustrate how micropatterns can facilitate cell-based assays. Alterations of the cell cytoskeleton are difficult to quantify in cells cultured on homogenous substrates, but culturing cells on micropatterns results in a reproducible organization of the actin meshwork due to systematic positioning of the cell adhesion contacts in every cell. Such normalization of the intracellular architecture allows quantification of even small effects on the actin cytoskeleton as demonstrated in these set of protocols using blebbistatin, an inhibitor of the actin-myosin interaction.

Adhesive micropatterns that normalize cellular architecture can be used to increase sensitivity in the detection of drug effects, improve reproducibility and simplify automated image acquisition and analysis. Such technology will benefit drug/siRNA screening assays, performed on conventional cell culture supports and consequently suffering from excessive cell-to-cell variability.

İlaç Etkisi Micropatterned Hücreler Geliştirilmiş Görselleştirme ve Kantitatif Analiz

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated ...

Biyomühendislik

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood cell (RBC) as adhesion sensor and presenting cell for one of the interacting molecules. It employs micromanipulation to bring the RBC into contact with another cell that expresses the other interacting molecule with precisely controlled area and time to enable bond formation. The adhesion event is detected as RBC elongation upon pulling the two cells apart. By controlling the density of the ligands immobilized on the RBC surface, the probability of adhesion is kept in mid-range between 0 and 1. The adhesion probability is estimated from the frequency of adhesion events in a sequence of repeated contact cycles between the two cells for a given contact time. Varying the contact time generates a binding curve. Fitting a probabilistic model for receptor-ligand reaction kinetics1 to the binding curve returns the 2D affinity and off-rate.
The assay has been validated using interactions of Fc&gamma; receptors with IgG Fc1-6, selectins with glycoconjugate ligands6-9, integrins with ligands10-13, homotypical cadherin binding14, T cell receptor and coreceptor with peptide-major histocompatibility complexes15-19.
The method has been used to quantify regulations of 2D kinetics by biophysical factors, such as the membrane microtopology5, membrane anchor2, molecular orientation and length6, carrier stiffness9, curvature20, and impingement force20, as well as biochemical factors, such as modulators of the cytoskeleton and membrane microenvironment where the interacting molecules reside and the surface organization of these molecules15,17,19.
The method has also been used to study the concurrent binding of dual receptor-ligand species3,4, and trimolecular interactions19 using a modified model21.
The major advantage of the method is that it allows study of receptors in their native membrane environment. The results could be very different from those obtained using purified receptors17. It also allows study of the receptor-ligand interactions in a sub-second timescale with temporal resolution well beyond the typical biochemical methods.
To illustrate the micropipette adhesion frequency method, we show kinetics measurement of intercellular adhesion molecule 1 (ICAM-1) functionalized on RBCs binding to integrin &alpha;L&beta;2 on neutrophils with dimeric E-selectin in the solution to activate &alpha;L&beta;2.

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

An adhesion frequency assay for measuring receptor-ligand interaction kinetics when both molecules are anchored on the surfaces of the interacting cells is described. This mechanically-based assay is exemplified using a micropipette-pressurized human red blood cell as adhesion sensor and integrin &alpha;L&beta;2 and intercellular adhesion molecule-1 as interacting receptors and ligands.

Yapışma için Frekans Testi Yerinde</emHücre-Hücre Arayüz Çapraz Junctional Moleküler Etkileşimler> Kinetik Analizi

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood ...

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and pathways as well as its ease of cultivation. Several C. elegans disease models have been reported, including neurodegenerative disorders such as Parkinson's disease (PD), which involves the degeneration of dopaminergic (DA) neurons 1. Both transgenes and neurotoxic chemicals have been used to induce DA neurodegeneration and consequent movement defects in worms, allowing for investigations into the basis of neurodegeneration and screens for neuroprotective genes and compounds 2,3.
Screens in lower eukaryotes like C. elegans provide an efficient and economical means to identify compounds and genes affecting neuronal signaling. Conventional screens are typically performed manually and scored by visual inspection; consequently, they are time-consuming and prone to human errors. Additionally, most focus on cellular level analysis while ignoring locomotion, which is an especially important parameter for movement disorders.
We have developed a novel microfluidic screening system (Figure 1) that controls and quantifies C. elegans' locomotion using electric field stimuli inside microchannels. We have shown that a Direct Current (DC) field can robustly induce on-demand locomotion towards the cathode ("electrotaxis") 4. Reversing the field's polarity causes the worm to quickly reverse its direction as well. We have also shown that defects in dopaminergic and other sensory neurons alter the swimming response 5. Therefore, abnormalities in neuronal signaling can be determined using locomotion as a read-out. The movement response can be accurately quantified using a range of parameters such as swimming speed, body bending frequency and reversal time.
Our work has revealed that the electrotactic response varies with age. Specifically, young adults respond to a lower range of electric fields and move faster compared to larvae 4. These findings led us to design a new microfluidic device to passively sort worms by age and phenotype 6.
We have also tested the response of worms to pulsed DC and Alternating Current (AC) electric fields. Pulsed DC fields of various duty cycles effectively generated electrotaxis in both C. elegans and its cousin C. briggsae 7. In another experiment, symmetrical AC fields with frequencies ranging from 1 Hz to 3 KHz immobilized worms inside the channel 8.
Implementation of the electric field in a microfluidic environment enables rapid and automated execution of the electrotaxis assay. This approach promises to facilitate high-throughput genetic and chemical screens for factors affecting neuronal function and viability.

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans&#039; Locomotion

A semi-automated micro-electro-fluidic method to induce on-demand locomotion in Caenorhabditis elegans is described. This method is based on the neurophysiologic phenomenon of worms responding to mild electric fields (&#x201C;electrotaxis&#x201D;) inside microfluidic channels. Microfluidic electrotaxis serves as a rapid, sensitive, low-cost, and scalable technique to screen for factors affecting neuronal health.

On-talep Kantitatif Analiz için mikroakışkan tabanlı Electrotaxis<em&gt; Caenorhabditis elegans</em&gt; &#39;Locomotion

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and ...

Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse and Parkinson&#39;s disease. Acute activation of G&#945;i/o-linked receptors inhibits AC activity, whereas persistent activation of these receptors results in heterologous sensitization of AC and increased levels of intracellular cAMP. Previous studies have demonstrated that this enhancement of AC responsiveness is observed both in vitro and in vivo following the chronic activation of several types of G&#945;i/o-linked receptors including D2 dopamine and &#956; opioid receptors. Although heterologous sensitization of AC was first reported four decades ago, the mechanism(s) that underlie this phenomenon remain largely unknown. The lack of mechanistic data presumably reflects the complexity involved with this adaptive response, suggesting that nonbiased approaches could aid in identifying the molecular pathways involved in heterologous sensitization of AC. Previous studies have implicated kinase and Gb&#947; signaling as overlapping components that regulate the heterologous sensitization of AC. To identify unique and additional overlapping targets associated with sensitization of AC, the development and validation of a scalable cAMP sensitization assay is required for greater throughput. Previous approaches to study sensitization are generally cumbersome involving continuous cell culture maintenance as well as a complex methodology for measuring cAMP accumulation that involves multiple wash steps. Thus, the development of a robust cell-based assay that can be used for high throughput screening (HTS) in a 384&#160;well format would facilitate future studies. Using two D2 dopamine receptor cellular models (i.e.&#160;CHO-D2L and HEK-AC6/D2L), we have converted our 48-well sensitization assay (&#62;20 steps 4-5 days) to a five-step, single day assay in 384-well format. This new format is amenable to small molecule screening, and we demonstrate that this assay design can also be readily used for reverse transfection of siRNA in anticipation of targeted siRNA library screening.

Persistent activation of inhibitory G protein-coupled receptors results in sensitization of adenylyl cyclase signaling. To identify the essential molecular pathways, nonbiased approaches are necessary; however, this strategy requires the development of a scalable cell-based cAMP sensitization assay. Herein, we describe a sensitization assay for small molecule and siRNA screening.

Adenilat siklaz uyuşturucu kaynaklı Sensitizasyonu: Küçük Molekül ve siRNA Tarama Uygulamaları için Deney düzene ve minyatür

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse ...

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range of 10-100 &#197;. By measuring the distances under various ligated conditions, conformational changes of the protein can be easily assessed. With LRET, a lanthanide, most often chelated terbium, is used as the donor fluorophore, affording advantages such as a longer donor-only emission lifetime, the flexibility to use multiple acceptor fluorophores, and the opportunity to detect sensitized acceptor emission as an easy way to measure energy transfer without the risk of also detecting donor-only signal. Here, we describe a method to use LRET on membrane proteins expressed and assayed on the surface of intact mammalian cells. We introduce a protease cleavage site between the LRET fluorophore pair. After obtaining the original LRET signal, cleavage at that site removes the specific LRET signal from the protein of interest allowing us to quantitatively subtract the background signal that remains after cleavage. This method allows for more physiologically relevant measurements to be made without the need for purification of protein.

We describe here an improved Luminescence Resonance Energy Transfer (LRET) method where we introduce a protease cleavage site between the donor and acceptor fluorophore sites. This modification allows us to obtain specific LRET signals arising from membrane proteins of interest, allowing for the study of membrane proteins without protein purification.

Lüminesans Rezonans Enerji memeli hücrelerinde ifade Membran Proteinleri Konformasyonel Değişiklikleri Eğitim transfer

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range ...

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen Presenting Cells (APCs). This is despite T-cell antigen receptors (TCRs) exerting usually a moderate affinity (&#181;M range) to antigen when binding is measured in vitro1. In view of the molecular and cellular parameters contributing to T-cell antigen sensitivity, a microscopy-based methodology has been developed as a means to monitor TCR-pMHC binding in situ, as it occurs within the synapse of a live T-cell and an artificial and functionalized glass-supported planar lipid bilayer (SLB), which mimics the cell membrane of an Antigen presenting Cell (APC) 2. Measurements are based on F&#246;rster Resonance Energy Transfer (FRET) between a blue- and red-shifted fluorescent dye attached to the TCR and the pMHC. Because the efficiency of FRET is inversely proportional to the sixth power of the inter-dye distance, one can employ FRET signals to visualize synaptic TCR-pMHC binding. The sensitive of the microscopy approach supports detection of single molecule FRET events. This allows to determine the affinity and off-rate of synaptic TCR-pMHC interactions and in turn to interpolate the on-rate of binding. Analogous assays could be applied to measure other receptor-ligand interactions in their native environment.

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

This manuscript describes how to conduct (single molecule) F&#246;rster Resonance Energy Transfer (FRET)- based assays to measure the binding dynamics between T-cell antigen receptor (TCR) and antigenic peptide-loaded MHC molecules as they occur within the immunological synapse of a T-cell in contact with a functionalized planar supported lipid bilayer.

TCR-pMHC Bağlama Ölçüm<em&gt; In Situ</em&gt; Bir FRET tabanlı Mikroskopi Assay kullanılarak

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen ...

Sensitive Detection of Proteopathic Seeding Activity with FRET Flow Cytometry

Increasing evidence supports transcellular propagation of toxic protein aggregates, or proteopathic seeds, as a mechanism for the initiation and progression of pathology in several neurodegenerative diseases, including Alzheimer's disease and the related tauopathies. The potentially critical role of tau seeds in disease progression strongly supports the need for a sensitive assay that readily detects seeding activity in biological samples. 
By combining the specificity of fluorescence resonance energy transfer (FRET), the sensitivity of flow cytometry, and the stability of a monoclonal cell line, an ultra-sensitive seeding assay has been engineered and is compatible with seed detection from recombinant or biological samples, including human and mouse brain homogenates. The assay employs monoclonal HEK 293T cells that stably express the aggregation-prone repeat domain (RD) of tau harboring the disease-associated P301S mutation fused to either CFP or YFP, which produce a FRET signal upon protein aggregation. The uptake of proteopathic tau seeds (but not other proteins) into the biosensor cells stimulates aggregation of RD-CFP and RD-YFP, and flow cytometry sensitively and quantitatively monitors this aggregation-induced FRET. The assay detects femtomolar concentrations (monomer equivalent) of recombinant tau seeds, has a dynamic range spanning three orders of magnitude, and is compatible with brain homogenates from tauopathy transgenic mice and human tauopathy subjects. With slight modifications, the assay can also detect seeding activity of other proteopathic seeds, such as &#945;-synuclein, and is also compatible with primary neuronal cultures. The ease, sensitivity, and broad applicability of FRET flow cytometry makes it useful to study a wide range of protein aggregation disorders.

Cell-to-cell transfer of protein aggregates, or proteopathic seeds, may underlie the progression of pathology in neurodegenerative diseases. Here, a novel FRET flow cytometry assay is described that enables specific and sensitive detection of seeding activity from recombinant or biological samples.

FRET Akım Sitometrisi ile Proteopathic Tohumculuk Faaliyet duyarlı Algılama

Increasing evidence supports transcellular propagation of toxic protein aggregates, or proteopathic seeds, as a mechanism for the initiation and ...

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

Damlacık Mikroakiskan için renkli Floresan Algılama Optik Elyaf kullanma

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

A Protocol for Using F&#246;rster Resonance Energy Transfer (FRET)-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex

The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. Nesprin-2G is a key component of the LINC complex that connects the actin cytoskeleton to membrane proteins (SUN domain proteins) in the perinuclear space. These membrane proteins connect to lamins inside the nucleus. Recently, a F&#246;rster Resonance Energy Transfer (FRET)-force probe was cloned into mini-Nesprin-2G (Nesprin-TS (tension sensor)) and used to measure tension across Nesprin-2G in live NIH3T3 fibroblasts. This paper describes the process of using Nesprin-TS to measure LINC complex forces in NIH3T3 fibroblasts. To extract FRET information from Nesprin-TS, an outline of how to spectrally unmix raw spectral images into acceptor and donor fluorescent channels is also presented. Using open-source software (ImageJ), images are pre-processed and transformed into ratiometric images. Finally, FRET data of Nesprin-TS is presented, along with strategies for how to compare data across different experimental groups.

A Protocol for Using F&amp;#246;rster Resonance Energy Transfer (FRET)-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex

A number of FRET-based force biosensors have recently been developed, enabling the protein-specific resolution of intracellular force. In this protocol, we demonstrate how one of these sensors, designed for the linker of the nucleoskeleton-cytoskeleton (LINC) complex protein Nesprin-2G can be used to measure actomyosin forces on the nuclear LINC complex.

Förster Rezonans Enerji Transferi (FRET) -force biyosensörlerin kullanılması için Protokol Nükleer LINC Kompleksi karşısında Mekanik Kuvvetleri Ölçmek için

The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. ...

Engineering Molecular Recognition with Bio-mimetic Polymers on Single Walled Carbon Nanotubes

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the optical window where biological samples are most transparent. Here, we outline techniques to adsorb amphiphilic polymers and polynucleic acids onto the surface of SWNTs to engineer their corona phases and create novel molecular sensors for small molecules and proteins. These functionalized SWNT sensors are both biocompatible and stable. Polymers are adsorbed onto the nanotube surface either by direct sonication of SWNTs and polymer or by suspending SWNTs using a surfactant followed by dialysis with polymer. The fluorescence emission, stability, and response of these sensors to target analytes are confirmed using absorbance and near-infrared fluorescence spectroscopy. Furthermore, we demonstrate surface immobilization of the sensors onto glass slides to enable single-molecule fluorescence microscopy to characterize polymer adsorption and analyte binding kinetics.

We present a protocol for engineering the corona phase of near infrared fluorescent single walled carbon nanotubes (SWNTs) using amphiphilic polymers and DNA to develop sensors for molecular targets without known recognition elements.

Tek Karbon Nanotüp Bio-mimetik Polimerler ile Mühendislik Moleküler Tanıma

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the ...