Name: a colorimetric assay that specifically measures granzyme b proteolytic activity: hydrolysis of boc-ala-ala-asp-s-bzl
Uploaded: 2014-11-28T15:00:00.000+00:00
Duration: 5 min 20 s
Description: Watch this Scientific Journal Video about A Colorimetric Assay that Specifically Measures Granzyme B Proteolytic Activity: Hydrolysis of Boc-Ala-Ala-Asp-S-Bzl at JoVE.com

This work received support through grant HA 6136/1-1 from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) to MH. JAT is supported by Program and Project Grants from the National Health and Medical Research Council of Australia.

NOT: farelerden elde edildi Dalaklar (yaş 6-10 hafta) ve tüm hayvan deneyleri Peter MacCallum Kanser Merkezi (E486) hayvan etiği kurallarına göre yapılmıştır. Örneklerin 1. hazırlanması. <ol><li> Aktif fare NK hücrelerinin hazırlanması. <ol><li> Ticari olarak temin edilebilen kitler kullanılarak negatif seçim ile (GzmB gen boş) fareler - / - C57BL / 6 ya da B6.GrzmB dalak tek hücre süspansiyonları, birincil saf NK hücrelerini izole edin. </li><li> Üreticilerin protokolünü izleyin. Kısaca, manyetik T ve B hücreleri, dendritik hücreler, granülositler, makrofajlar ve spesifik yüzey belirteçleri karşı biyotin-konjuge antikor ile kırmızı kan hücreleri gibi &quot;NK-olmayan&quot; hücreleri etiketlemek. &quot;Non-NK&quot; hücreleri tüketmek anti-Biyotin boncuklar ile manyetik ayırma kullanın. </li><li> Kültür, 24 oyuklu plakalar içinde 700,000 hücre / ml IL-2 (1000 U / ml) ihtiva eden ortam içinde 5-7 gün için, NK hücreleri izole37 ° C&#39;de &#160; ° C. </li><li> Seçenek olarak ise, kullanım 19 T-hücreleri, OT1 T hücresi reseptörü transgenik fareler 20 birincil antijen sınırlı CTL CTL salgılanan GzmB 19 aktivitesini belirlemek için aktif NK hücrelerinden karma lenfosit kültürleri 21 ya da yüzer üretilen aktif. </li></ol></li><li> Insan NK hücre hattı ve birinci NK hücrelerinin izolasyonu bakımı. <ol><li> % 10 cenin dana serumu ile takviye edilmiş Roswell Park Memorial Institute (RPMI) ortamında insan NK lösemi (YT hücre) koruyun. Seçenek olarak ise, sağlıklı bireylerden alınan periferal kanda, primer, NK hücrelerinin saflaştırılması ve daha önce tarif edildiği gibi 22, IL-2, 100 U / ml sonda yoğunluğunda içeren bir ortam içinde 2-3 gün için harekete geçirir. </li></ol></li><li> Hücre lizatlarının üretilmesi. <ol><li> PBS (pH 7.4) içinde hücreleri üç kez yıkayın, sonra Nonidet P-40 liziz tamponunda askıya (% 0.1 NP-40, 250 mM NaCI, 25 mM Hepes, 2.5 mM EDTA). İdeal olarak, 5 x 10 6, en az lize </sNK hücreleri, ya da 100 ul tampon 1 x 10 7 T hücreleri&gt; kadar. Proteaz inhibitörleri ekleme, birçok gibi GzmA gibi geri dönüşü olmayan bir serin proteaz inhibitörleri ve özellikle tryptases içindedir. 20 dakika boyunca buz üzerinde inkübe edilir ve daha sonra 10 dakika boyunca 15.000 x g&#39;de bir mikrosantrfuj çekirdekleri pelet. Nükleer pelet atın ve yeni bir tüp süpernatant aktarın. </li></ol></li><li> Lisatların protein konsantrasyonu belirlenir. <ol><li> Örneğin Bradford veya bisinkoninik asit (BCA) olarak tercih edilen ticari olarak temin edilebilen bir protokol kullanarak protein konsantrasyonunu belirleyin. Konsantrasyonu ~ 2 mg / ml az olduğundan emin olun. Gerekene kadar -20 ° C&#39;de saklayın lisatları (granzim aktivitesi, -20 ° C&#39;de birkaç ay boyunca stabildir). </li></ol></li></ol> 2. Granzim B Aktivite Deneyi <ol><li> Reaktiflerin hazırlanması <ol><li> DMSO içinde substratların (Boc-AAD-S-Bzl veya Ac-IEPD-pNA) içindeki bir 10mM stok hazırlayın (~ 5 mg / ml) -20 &amp; # &#39;de parçalar halinde ve mağaza176 ° C. Taze bir çalışma seyreltme hazırlayın ve Boc-AAD-S-Bzl kısmen DMSO içinde depolama hidrolize olarak, her gün sonra atın. </li><li> -20 ° C&#39;de, 250 mM DMSO içinde kolorimetrik reaktif (5,5&#39;-ditiyo-bis (2-nitrobenzoik asit), DTNB) stok (0.09 g / ml) ve mağaza hazırlayın. Her gün taze bir çalışma seyreltme hazırlayın. Bu reaktif sadece SBzl gösterge değil, gruplar pNA alt tabakalar için gereklidir. </li><li> Taze tampon (0.1 M HEPES, 0.05 M MgCl2, pH 7.3) her 2-3 hafta sonra yapılır. </li></ol></li><li> Oda sıcaklığına DTNB ve sentetik yüzeylerde getirin. Seyreltik alt-tabakalar (1:16, örneğin, 1.6 ml tampon içinde 100 | il) ve DTNB (1: 250, örneğin 10 2.5 mi tampon maddesi içinde ul) ve iyice karıştırın. </li><li> 160 ul son hacim olması için tampon maddesi içinde her bir numunenin 100 ug protein lizat az seyreltin. Bir çok kanallı pipet kullanarak bir &quot;&#39;U&#39; alt vinil 96 oyuklu plaka (~ 33.3 ug / çukur), üç kopya halinde oyuk başına 50 ul ekle.Bir &quot;tampon sadece&quot; kontrol dahil (ayrıca üç kopya halinde). </li><li> Ac-IEPD-pNA ile çalışırken örnekleri / tampon kontrolüne seyreltilmiş DTNB 100 ul ekleyin (bu adımı atlayabilirsiniz. NOT: GzmB tarafından Ac-IEPD-pNA&#39;nın yarılması doğrudan fluorojenik pNA (Şekil 4B) serbest bırakır. </li><li> Sonra hızlı bir şekilde tampon herhangi bir olumsuz kontroller ile başlayan ve beklenen olumlu örnekleri ile terbiye, üçlü her set için seyreltilmiş yüzeylerde 50 ul eklemek için bir çok kanallı pipet kullanın. </li><li> Plaka okuyucu plaka koyun. OD 405 nm&#39;de bir mikro-plaka okuyucusu içinde Boc-Ala-Ala-Asp-S-Bzl / Ac-IEPD-pNA hidrolizi ile Asp-az aktivitesi ölçülür. 25 okuma, her 15 sn (yaklaşık 6 dk, toplam okuma süresi) almak, bir kinetik tahlil protokolü kullanın. </li><li> Plaka okuyucu yazılım Vmax için bir değer oluşturmak değil eğer elle substrat bölünme oranını hesaplamak için her zaman noktasında oluşturulan absorbans okumaları kullanın. </li></ol> Da 3. Analizta NOT: Zamanla OD değişikliği (mOD / dk) olarak tanımlanan maksimum hız olarak sunulmaktadır üretilen veriler,. <ol><li> Verileri analiz etmek için, absorbans değişikliği oranı hesaplanır alt-tabaka bölünme maksimum hızını belirler. Otomatik genellikle bu seçeneği vardır plaka okuyucu yazılımı kullanarak yapın. </li><li> Alternatif elle, plaka okuyucu üzerinde kinetik tahlil sırasında oluşturulan absorbans değişim, kinetik araziler görüntüledikten sonra dik düz bir çizgi verir veri noktalarını seçin. En deneyleri için maksimum hız tahlilin ilk 2 dakika içinde elde edilmektedir. </li><li> Zaman aralığı bu düz çizgi ve bölünmenin en yüksek OD okuma başlangıç ​​OD okuma çıkartın. İstatistiksel analiz ve grafik ekran için Microsoft Excel / Graph pad ham veri aktarın. </li></ol>

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The authors have nothing to disclose.

Tarihsel olarak granzymes hedef hücreler hızlı bir apoptotik ölümünü indükleyebilen sitotoksik lenfositleri (CTL ve NK hücreleri) anahtar etkeni moleküller olarak tespit edilmiştir. Bu, aspartat (D) artıklarının hedef alt-tabaka molekülleri ayrılır ve böylece aşağı doğru hedeflerin çeşitli yanı sıra her ikisi de yaran ön-kaspaz kaspaz kaskadını aktive mümkün GzmB aksiyonu için esas oldu. Bununla birlikte, şu anda GzmB sentezleme lenfoid sitotoksik hücreler ile sınırlı değildir ve bu...

Granzymes doğal katil (NK) hücreler ve sitotoksik T lenfositler (CTL), 1 salgılama lizozomlarında bulunan serin proteaz ailesidir. Beş farklı granzymes insanlarda (A, B, H, K ve M,) &#39;de bulunan, ve farelerde on (A - G, K, M ve N) 2,3. Granzim A ve Granzim B (GzmA, GzmB) en bol miktarda bulunan ve geniş ölçüde insan ve kemirgen ortamda araştırılmıştır. GzmB klasik fonksiyonu hedef hücre sitosol 4 ulaşmak için Granzim izin gözenek oluşturucu protein perforin ile bağlantılı olarak yürütülen, hedef hücrelerde apoptoz endüksiyonu. GzmB sentezleme tümden sitotoksik lenfositlerinin de bulunmasına rağmen, son zamanlarda yapılan çalışmalar bazofiller 6, mast hücreleri 7, plazmasitoid dendritik hücreler 8 ve B hücreleri 9, aşağıdakileri içeren, ancak keratinositler 5 ile sınırlı değildir, başka bir GzmB ifade eden hücre tipleri, çeşitli ele edilmiştir, 10.Bu bağlamda, apoptotik olmayan GzmB fonksiyonları, enflamatuar süreçler, doku tekrar modellemesinde ve diğer bağışıklık düzenleyici özelliklerinin 11-14 katılım kadar ortaya çıkmıştır. Daha geniş bir biyolojik rolü, daha önce şüphe daha GzmB önerilmiştir göz önüne alındığında, araştırmacılar da tespiti için güvenilir ve özel araçlar gerektirir. Avantajlı olarak ökaryotik serin proteazları 15 arasında benzersiz aspartik asit kalıntısı, bir özelliği karboksil yanı üzerinde bölmek için GzmB spesifik bir gerekliliktir. Fare, insan ve sıçan GzmB ancak fare GzmB uzun alt-tabaka özgüllüğü terminali (P1) Asp, bazı genel alt-tabakalar GzmB tarafından etkin bir şekilde klivaj edilebilir olduğu anlamına gelir, hem insan ve fare 16, bu ustaca farklıdır, yapısal olarak çok benzer Üç türlerinden, üst baş P1 daha karmaşık dizileri ile diğer alt-tabakalara ise, geniş çapta değişkendir sonuçlar verebilir. Geçmişte ve daha yeni li hem deterature, bu gerçeği dikkatle kontrol olsa, önemli karışıklık ve bazı deneysel bulguların biyolojik önemi yanlış yorumlanmasına neden oldu, kinetik çalışmalar durumu 17 düzeltmek için çalışmışlardır. Bu yazıda, iki ticari olarak temin edilebilen alt tabakalar, yani, Boc-Ala-Ala-Asp-SBzl ve N-asetil-Ile-Glu-Pro-Asp-p-nitroanilid kullanılarak, bu noktaları göstermek için çalışmışlardır. İki reaktifler bölünme (bir floresan serbest paranitroanilide karşı serbest sülfidril) Aşağıdaki farklı reaktif gruplar oluşturmak, ama bu proteolitik bölünme üzerinde hiçbir etkisi ne olursa olsun sahip. Açıklanan protokol, çok eski bir protokol 18 modern bir uyarlamasıdır, fakat, aynı zamanda, GzmA ve GzmH gibi diğer granzymes aktivitesini tespit etmek için bir yöntem çerçeve sağlarken araştırmacılar, uygun farklı GzmB alt tabakaları kullanır yardımcı olacaktır.

<table><tbody><tr><td>Boc-Ala-Ala-Asp-S-Bzl</td><td>SM Biochemicals LLC, CA</td><td>SMSB05</td><td>Granzyme B substrate (mouse and human)</td></tr><tr><td>Ac-IEPD-pNA&amp;nbsp;</td><td>SM Biochemicals LLC, CA</td><td>SMPNA009</td><td>Granzyme B substrate (only human)</td></tr><tr><td>N-&amp;#945;-CBZ-L-lysine-S-Bzl</td><td>Sigma-Aldrich</td><td>C3647</td><td>Granzyme A substrate</td></tr><tr><td>Suc-Phe-Leu-Phe-S-Bzl</td><td>SM Biochemicals LLC, CA</td><td>SB025</td><td>Granzyme H substrate</td></tr><tr><td>5,5&amp;rsquo;-dithio-bis(2-nitrobenzoic acid)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>D8130</td><td>DTNB, Ellman&amp;rsquo;s Reagent</td></tr><tr><td>NK cell isolation kit II mouse</td><td>Miltenyi Biotec GmbH</td><td>130-096-892</td><td>negative selection kit</td></tr><tr><td>NK cell isolation kit human</td><td>Miltenyi Biotec GmbH</td><td>130-092-657</td><td>negative selection kit</td></tr><tr><td>Plate reader</td><td>Biorad iMark</td><td></td><td>Biorad Microplate Manager Software Version MPM6.3</td></tr><tr><td>Serocluster U-bottom vinyl 96-well plate</td><td>Corning, MA, USA</td><td>2797</td><td></td></tr></tbody></table>

a colorimetric assay that specifically measures granzyme b proteolytic activity: hydrolysis of boc-ala-ala-asp-s-bzl

The serine protease Granzyme B (GzmB) mediates target cell apoptosis when released by cytotoxic T lymphocytes (CTL) or natural killer (NK) cells. GzmB is the most studied granzyme in humans and mice and therefore, researchers need specific and reliable tools to study its function and role in pathophysiology. This especially necessitates assays that do not recognize proteases such as caspases or other granzymes that are structurally or functionally related. Here, we apply GzmB&#8217;s preference for cleavage after aspartic acid residues in a colorimetric assay using the peptide thioester Boc-Ala-Ala-Asp-S-Bzl. GzmB is the only mammalian serine protease capable of cleaving this substrate. The substrate is cleaved with similar efficiency by human, mouse and rat GzmB, a property not shared by other commercially available peptide substrates, even some that are advertised as being suitable for this purpose. This protocol is demonstrated using unfractionated lysates from activated NK cells or CTL and is also suitable for recombinant proteases generated in a variety of prokaryotic and eukaryotic systems, provided the correct controls are used. This assay is a highly specific method to ascertain the potential pro-apoptotic activity of cytotoxic molecules in mammalian lymphocytes, and of their recombinant counterparts expressed by a variety of methodologies.

Boc-AAD-S-Bzl, alt-tabaka GzmB için spesifiktir serin proteaz GzmB sitotoksik lenfositleri (CTL ve NK hücreleri) önemli bir bileşeni olan ve bu tip virüs ile enfekte olmuş veya dönüştürülmüş hücreler gibi hedef hücrelerin hızlı apoptotik ölümünü teşvik etmek için ağırlıklı olarak sorumludur. Bu durum, seçilen proteinler bazı özel aspartat kalıntısı sonra yarılma için alt-tabaka tercihine büyük ölçüde, bir öznitelik de aspartat kalınt...

Watch this Scientific Journal Video about A Colorimetric Assay that Specifically Measures Granzyme B Proteolytic Activity: Hydrolysis of Boc-Ala-Ala-Asp-S-Bzl at JoVE.com

A Colorimetric Assay that Specifically Measures Granzyme B Proteolytic Activity: Hydrolysis of Boc-Ala-Ala-Asp-S-Bzl

We describe a simple, quantitative colorimetric assay that specifically measures the proteolytic activity of human, mouse or rat Granzyme B (GzmB). This protocol can be easily adapted for determining protease activity of other granule serine proteases by the hydrolysis of other synthetic peptide substrates with an appropriate recognition sequence.

Boc-Ala-Ala-Asp-S-Bzl hidrolizi, A Kolorimetrik Özellikle Granzim B proteolitik aktivitesini ölçer Deneyi

The serine protease Granzyme B (GzmB) mediates target cell apoptosis when released by cytotoxic T lymphocytes (CTL) or natural killer (NK) cells. GzmB is ...

a-colorimetric-assay-that-specifically-measures-granzyme-b

Universidad de Cartagena UDEC

Research

JoVE Journal

Chemistry

13.0K Görüntüleme.  Peter MacCallum Cancer Centre. We describe a simple, quantitative colorimetric assay that specifically measures the proteolytic activity of human, mouse or rat Granzyme B (GzmB). This protocol can be easily adapted for determining protease activity of other granule serine proteases by the hydrolysis of other synthetic peptide substrates with an appropriate recognition sequence.

Video: A Colorimetric Assay that Specifically Measures Granzyme B Proteolytic Activity: Hydrolysis of Boc-Ala-Ala-Asp-S-Bzl

Detection of Protease Activity by Fluorescent Peptide Zymography

The purpose of this method is to measure the proteolytic activity of complex biological samples. The samples are separated by molecular weight using electrophoresis through a resolving gel embedded with a degradable substrate. This method differs from traditional gel zymography in that a quenched fluorogenic peptide is covalently incorporated into the resolving gel instead of full length proteins, such as gelatin or casein. Use of the fluorogenic peptides enables direct detection of proteolytic activity without additional staining steps. Enzymes within the biological samples cleave the quenched fluorogenic peptide, resulting in an increase in fluorescence. The fluorescent signal in the gels is then imaged with a standard fluorescent gel scanner and quantified using densitometry. The use of peptides as the degradable substrate greatly expands the possible proteases detectable with zymographic techniques.

Here, we present a detailed protocol for a modified zymographic technique in which fluorescent peptides are used as the degradable substrate in place of native proteins. Electrophoresis of biological samples in fluorescent peptide zymograms enables detection of a wider range of proteases than previous zymographic techniques.

Floresan peptid Zymography tarafından proteaz aktivitesinin algılama

The purpose of this method is to measure the proteolytic activity of complex biological samples. The samples are separated by molecular weight using ...

Kanser Araştırmaları

Measuring Composition of CD95 Death-Inducing Signaling Complex and Processing of Procaspase-8 in this Complex

Extrinsic apoptosis is mediated by the activation of death receptors (DRs) such as CD95/Fas/APO-1 or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-receptor 1/receptor 2 (TRAIL-R1/R2). Stimulation of these receptors with their cognate ligands leads to the assembly of the death-inducing signaling complex (DISC). DISC comprises DR, the adaptor protein Fas-associated protein with death domain (FADD), procaspases-8/-10, and cellular FADD-like interleukin (IL)-1&#946;-converting enzyme-inhibitory proteins (c-FLIPs). The DISC serves as a platform for procaspase-8 processing and activation. The latter occurs via its dimerization/oligomerization in the death effector domain (DED) filaments assembled at the DISC. 
Activation of procaspase-8 is followed by its processing, which occurs in several steps. In this work, an established experimental workflow is described that allows the measurement of DISC formation and the processing of procaspase-8 in this complex. The workflow is based on immunoprecipitation techniques supported by western blot analysis. This workflow allows careful monitoring of different steps of procaspase-8 recruitment to the DISC and its processing and is highly relevant for investigating molecular mechanisms of extrinsic apoptosis.

Here, an experimental workflow is presented that enables the detection of caspase-8 processing directly at the death-inducing signaling complex (DISC) and determines the composition of this complex. This methodology has broad applications, from unraveling the molecular mechanisms of cell death pathways to the dynamic modeling of apoptosis networks.

CD95 Ölüme Neden Olan SinyalIzasyon Kompleksinin Bileşiminin Ölçülmesi ve Procaspase-8'in Bu Komplekste İşleniş

Extrinsic apoptosis is mediated by the activation of death receptors (DRs) such as CD95/Fas/APO-1 or tumor necrosis factor-related apoptosis-inducing ...

Biyokimya

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

Nitel ve Nicel Saptanması için Tahliller ve Protein Antimikrobiyal karakterizasyonu

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

İmmünoloji ve Enfeksiyon

Measuring Caspase Activity Using a Fluorometric Assay or Flow Cytometry

The activation of cysteine proteases, known as caspases, remains an important process in multiple forms of cell death. Caspases are critical initiators and executioners of apoptosis, the most studied form of programmed cell death. Apoptosis occurs during developmental processes and is a necessary event in tissue homeostasis. Pyroptosis is another form of cell death that utilizes caspases and is a critical process in activating the immune system through the activation of the inflammasome, which results in the release of members of the interleukin-1 (IL-1) family. To assess caspase activity, target substrates can be assessed. However, sensitivity can be an issue when examining single cells or low-level activity. We demonstrate how a fluorogenic substrate can be used with a population-based assay or single-cell assay by flow cytometry. With proper controls, different amino acid sequences can be used to identify which caspases are active. Using these assays, the simultaneous loss of the inhibitors of apoptosis proteins upon tumor necrosis factor (TNF) stimulation has been identified, which primarily induces apoptosis in macrophages rather than other forms of cell death.

The present protocol describes two methods to measure caspase activity through a fluorogenic substrate using flow cytometry or a spectrofluorometer.

Florometrik Tahlil veya Akış Sitometrisi Kullanarak Kaspaz Aktivitesinin Ölçülmesi

The activation of cysteine proteases, known as caspases, remains an important process in multiple forms of cell death. Caspases are critical initiators ...

PROTAC'larda Üçlü Kompleks Oluşumunun Biyofiziksel Deneyler Kullanılarak Karakterizasyonu

The Development and Application of Biophysical Assays for Evaluating Ternary Complex Formation Induced by Proteolysis Targeting Chimeras (PROTACS)

E3 ligases and proteins targeted for degradation can be induced to form complexes by heterobifunctional molecules in a multi-step process. The kinetics and thermodynamics of the interactions involved contribute to efficiency of ubiquitination and resulting degradation of the protein. Biophysical techniques such as surface plasmon resonance (SPR), biolayer interferometry (BLI), and isothermal titration calorimetry (ITC) provide valuable information that can be used in the optimization of those interactions. Using two model systems, a biophysical assay tool kit for understanding the cooperativity of ternary complex formation and the impact of the &#39;hook effect&#39; on binding kinetics was established. In one case, a proteolysis targeting chimera (PROTAC) molecule that induced ternary complex formation between Brd4BD2 and VHL was evaluated. The heterobifunctional molecule, MZ1, has nM affinities for both the Brd4BD2 protein (SPR KD = 1 nM, ITC KD = 4 nM) and the VHL complex (SPR KD = 29 nM, ITC KD = 66 nM). For this system, robust SPR, BLI, and ITC assays were developed that reproduced published results demonstrating the cooperativity of ternary complex formation. In the other case, a molecule that induced ternary complexes between a 46.0 kDa protein, PPM1D, and cereblon [CRBN (319-442)] was studied. The heterobifunctional molecule, BRD-5110, has an SPR KD = 1 nM for PPM1D but much weaker binding against the truncated CRBN (319-442) complex (SPR KD= ~ 3 &#181;M). In that case, the binding for CRBN in SPR was not saturable, resulting in a &#34;hook-effect&#34;. Throughput and reagent requirements for SPR, BLI, and ITC were evaluated, and general recommendations for their application to PROTAC projects were provided.

Yazar Spotlight: PROTACS Üçlü Komplekslerini Karakterize Etmek için Biyofiziksel Tahlillerin Değerlendirilmesi 

Here we describe protocols for the biophysical characterization of ternary complex formation induced by proteolysis targeting chimeras (PROTACS) that involve the ubiquitin ligases Von Hippel-Lindau E3 ligase (VHL) and Cereblon (CRBN). Biophysical methods illustrated herein include surface plasmon resonance (SPR), biolayer interferometry (BLI), and isothermal titration calorimetry (ITC).

Kimeraları Hedefleyen Proteoliz Tarafından İndüklenen Üçlü Kompleks Oluşumunun Değerlendirilmesi için Biyofiziksel Testlerin Geliştirilmesi ve Uygulanması (PROTACS)

E3 ligases and proteins targeted for degradation can be induced to form complexes by heterobifunctional molecules in a multi-step process. The kinetics ...

<meta charset="utf8"></head><body>Elastaz Modülasyonunun Ölçülmesi: Terapötik İçgörüler için Kolorimetrik Bir Test

Quantifying the Modulation of Elastase Enzyme Activity Through Colorimetric Analysis

Elastase, a serine protease, plays an essential role in elastin degradation. Elastin is an extracellular protein that helps maintain tissue elasticity in the lungs, skin, and blood vessels. Tight regulation of elastase activity is crucial for tissue homeostasis, as dysregulation can contribute to pathologies such as emphysema, wrinkles, and atherosclerosis. Some compounds, such as naturally occurring phytochemicals, have shown potential for therapeutic intervention and have attracted significant interest. Elucidating the modulatory effects of different compounds on elastase, whether inhibitory or stimulatory, is crucial for developing novel therapeutic and cosmetic strategies targeting elastase-associated disorders. A widely accepted method for measuring elastase activity is the colorimetric elastase assay. In this assay, a specific substrate is used to break down elastase, releasing a detectable yellow compound, p-nitroaniline (pNA). The amount of pNA produced reflects elastase activity in the sample and can be measured by colorimetry. This assay offers several benefits, including simplicity, high sensitivity, rapid results, and adaptability to various research needs. The colorimetric elastase assay remains a valuable tool for studying how compounds impact elastase activity. Due to its ease of use and effectiveness, this assay is a cornerstone of research in this field.

<meta charset="utf8"></head><body>Kolorimetrik Analiz ile Elastaz Enzim Aktivitesinin Modülasyonunun Ölçülmesi

This protocol describes the development of a colorimetric assay method for determining the ability of compounds to inhibit or activate elastase activity.

Kolorimetrik Analiz ile Elastaz Enzim Aktivitesinin Modülasyonunun Ölçülmesi

Elastase, a serine protease, plays an essential role in elastin degradation. Elastin is an extracellular protein that helps maintain tissue elasticity in ...

Boc-Ala-Ala-Asp-S-Bzl hidrolizi, A Kolorimetrik Özellikle Granzim B proteolitik aktivitesini ölçer Deneyi

Özet

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Bu videodaki bölümler

Floresan peptid Zymography tarafından proteaz aktivitesinin algılama

CD95 Ölüme Neden Olan SinyalIzasyon Kompleksinin Bileşiminin Ölçülmesi ve Procaspase-8'in Bu Komplekste İşleniş

Nitel ve Nicel Saptanması için Tahliller ve Protein Antimikrobiyal karakterizasyonu

Florometrik Tahlil veya Akış Sitometrisi Kullanarak Kaspaz Aktivitesinin Ölçülmesi

Kimeraları Hedefleyen Proteoliz Tarafından İndüklenen Üçlü Kompleks Oluşumunun Değerlendirilmesi için Biyofiziksel Testlerin Geliştirilmesi ve Uygulanması (PROTACS)

Kimeraları Hedefleyen Proteoliz Tarafından İndüklenen Üçlü Kompleks Oluşumunun Değerlendirilmesi için Biyofiziksel Testlerin Geliştirilmesi ve Uygulanması (PROTACS)

Kimeraları Hedefleyen Proteoliz Tarafından İndüklenen Üçlü Kompleks Oluşumunun Değerlendirilmesi için Biyofiziksel Testlerin Geliştirilmesi ve Uygulanması (PROTACS)

Kolorimetrik Analiz ile Elastaz Enzim Aktivitesinin Modülasyonunun Ölçülmesi

Kolorimetrik Analiz ile Elastaz Enzim Aktivitesinin Modülasyonunun Ölçülmesi

Kolorimetrik Analiz ile Elastaz Enzim Aktivitesinin Modülasyonunun Ölçülmesi