أشكر لك الدكتور برافين كومار في المختبر ويتمان في سان فرانسيسكو للفيلم من الأكتين في الخلايا HaCAT.

1. إعداد أكتين وكان مصدر أكتين المستخدمة في هذا الاختبار غير العضلات الصفائح الدموية البشرية (β - أكتين) تم الحصول عليها من أسهم شركة aliquots الهيكل الخلوي في 10 يتم الاحتفاظ ملغ / مل في الثلاجة -70 درجة مئوية. للفحص ، هو المخفف للأكتين إلى 0.4 ملغ / مل في منطقة عازلة تحتوي على 5 ملم تريس الرقم الهيدروجيني 8 ، 0.2 ملم CaCl 2 ، 0.2 و 0.5 ملي ATP DTT ملم ، في طرد 20000 لمدة 10 دقيقة ز عند 4 درجة مئوية. وطاف ، وهو أكتين موحودي جاهزة للبلمرة. هي التي يسببها أكتين البلمرة من بوكل إضافة 50 مم ، و 1 و 2 ATP ملي ملي MgCl 2. وتحدث البلمرة في درجة حرارة الغرفة لمدة 1 ساعة. 2. التحضير للبروتين في هذا الاختبار ، ونحن باستخدام C - محطة من تالين ، الذي كان بمثابة تنقية البروتين GST الموسومة. يخضع هذا البروتين لاستخدامها في مقايسة إلى الطرد المركزي عالية السرعة لمسح ما قبل المجاميع قبل الحضانة مع أكتين - F. هو نسج من البروتين في 100000 ز في نابذة فائقة السرعة لمدة 20 دقيقة بيكمان في 22 درجة مئوية. 3. أكتين البروتين الحضانة فإن كمية الأكتين والبروتين في مقايسة تختلف. في هذا المثال نحن نستخدم أكتين في الزائدة. عادة ، يتم حساب نسبة المولي ، على سبيل المثال. نسبة 4:01 من المولي لأكتين البروتين. في مقايسة التالية ، وحجم رد الفعل النهائي هو 150 ميكرولتر. سوف العازلة التي يتم تحضين البروتين و F - أكتين تعتمد على البروتين لفحصها ، وعلى الشروط المطلوبة. في هذا المثال ، نستخدم العازلة التي تحتوي على 10 ملي تريس الرقم الهيدروجيني 7.0 ، 1 ATP ملي و 0.2 ملي DTT ، 1 EGTA ملم ، 0.1 ملم CaCl 2 و 2 مم MgCl 2. يتم تحضين البروتين و F - أكتين في المنطقة العازلة لمدة 1 ساعة في درجة حرارة الغرفة. 4. الترسيب من طراز F - أكتين بعد الحضانة ، ونسج على عينات 100000 ز في 22 درجة مئوية. نستخدم نابذة فائقة السرعة بيكمان في الفيديو. تتم إزالة الدوار بعناية حتى لا تعكر صفو الكريات. 5. تحليل البروتين المشترك الترسيب مع F - أكتين تتم إزالة بعناية من قبل supernatants pipetting في أنبوب إيبندورف و 5 X Laemmli العازلة عينة SDS - PAGE يضاف. يضاف إلى حجم مناسب من 1 العازلة Laemmli X عينة SDS - PAGE إلى الكريات المتبقية ، pipetted صعودا وهبوطا ، ونقل إلى أنابيب إيبندورف جديدة. ويتم تحليل الكميات النسبية للبروتين في كريات supernatants وبعد الانفصال من جانب وSDS - PAGE Coomassie الأزرق تلطيخ من هلام أو النشاف الغربية. ويمكن من أجل تحديد خصوصية التفاعل مع أكتين البروتين ، يمكن تنفيذ الأكتين التي تعتمد على تركيز التعاون الترسبات. يتم تحضين لهذا النوع من التجربة ، وهو مبلغ ثابت من البروتين ، وسلسلة من كميات متزايدة من الأكتين ، والذي أجري على النحو المبين أعلاه.

<ol>
	<li>Senetar, M. A., Foster, S. J., McCann, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrasteric+inhibition+mediates+the+interaction+of+the+I/LWEQ+module+proteins+Talin1,+Talin2,+Hip1,+and+Hip12+with+actin.">Intrasteric inhibition mediates the interaction of the I/LWEQ module proteins Talin1, Talin2, Hip1, and Hip12 with actin.</a> Biochemistry. 14, 15418-15428 (2004).</li><li>Goldmann, W. H., Hess, D., Isenberg, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+intact+talin+and+talin+tail+fragment+on+actin+filament+dynamics+and+structure+depends+on+pH+and+ionic+strength.">The effect of intact talin and talin tail fragment on actin filament dynamics and structure depends on pH and ionic strength.</a> Eur J Biochem. 260, 439-445 (1999).</li><li>Lee, H., Bellin, R. M., Walker, D. L., Patel, B., Powers, P., Liu, H., Garcia-Alvarez, B., de Pereda, J., Liddington, R. C., Volkmann, N., Hanein, D., Critchley, D. R., Robson, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+an+Actin-binding+Site+within+the+Talin+FERM+Domain.">Characterization of an Actin-binding Site within the Talin FERM Domain.</a> J Mol Biol. 22, 771-784 (2004).</li></ol>

أكتين المشترك الترسيب هو بسيط في فحص المختبر لتحليل البروتينات specfic ملزمة لأكتين - F. في هذا الفيديو ، ونظهر مثال واحد بسيط كيف أكتين المشترك الترسيب يمكن الاضطلاع بها. نعرض كيفية تحضير F - أكتين ، وإعداد هذا البروتين لفحصها وإجراء أكتين المشترك الترسيب. وينبغي النظر في عدد من النق...

actin co-sedimentation assay; for the analysis of protein binding to f-actin

الهيكل الخلوي أكتين داخل الخلية هو عبارة عن شبكة من خيوط الأكتين الذي يسمح للحركة الخلايا والعمليات الخلوية ، والتي تولد التوتر ويساعد في شكل يحافظ الخلوية. على الرغم من أن الهيكل الخلوي أكتين هو بنية جامدة ، بل هو بنية ديناميكية التي يتم إعادة عرض باستمرار. ويمكن لعدد من البروتينات ربط الهيكل الخلوي أكتين. هو المطلوب في كثير من الأحيان ملزمة من بروتين خاص لأكتين - F لدعم الملاحظات الخلية أو البيولوجية لتعزيز فهم العمليات الحيوية نتيجة لإعادة تشكيل الهيكل الخلوي أكتين. شارك في فحص الترسيب أكتين هو المختبر في فحص روتيني لتحليل الملزمة للبروتينات معينة أو المجالات البروتين مع أكتين - F. المبادئ الأساسية للمقايسة تنطوي على حضانة للبروتين من الفائدة (الطول الكامل أو مجال) مع F - أكتين ، خطوة تنبيذ فائق لتكوير F - أكتين وتحليل البروتين المترسبة التعاون مع أكتين - F. ويمكن تصميم أكتين المشترك الترسيب المقايسات وفقا لقياس أكتين الانتماءات الملزمة والمقايسات في المنافسة.

Watch this Scientific Journal Video about Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin at JoVE.com

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

البروتينات ربط أكتين الخيطية (F - أكتين) من خلال وحدات أكتين متميزة ملزمة. في هذا الفيديو نبدي الداخلي أكتين شارك في الترسيب ، وهو المختبر في فحص روتيني لتحليل البروتينات أو المجالات المحددة التي تربط F - أكتين.

أكتين المشارك الترسيب الفحص ، وبالنسبة للتحليل البروتين ملزمة لF - أكتين

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

actin-co-sedimentation-assay-for-analysis-protein-binding-to-f

Research

JoVE Journal

Biology

19.3K Views.  University of California, San Francisco - UCSF. البروتينات ربط أكتين الخيطية (F - أكتين) من خلال وحدات أكتين متميزة ملزمة. في هذا الفيديو نبدي الداخلي أكتين شارك في الترسيب ، وهو المختبر في فحص روتيني لتحليل البروتينات أو المجالات المحددة التي تربط F - أكتين.

Video: Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

والخلايا العصبية ونجمية المشارك الثقافة الفحص لتحليل محتوى عال من العصبية

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

اكتشاف بروتين Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique advantage of FSM is its ability to capture the movement and turnover kinetics (assembly/disassembly) of the F-actin network within living cells. This technique is particularly useful in deriving quantitative measurements of F-actin dynamics when paired with computer vision software (qFSM).  We describe  the selection, microinjection and visualization of fluorescent actin probes in living cells. Importantly, similar procedures are applicable to visualizing other macomolecular assemblies. FSM has been demonstrated for microtubules, intermediate filaments, and adhesion complexes.  

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM

Selection, microinjection, and imaging of fluorescently-labeled F-actin via fluorescent speckle microscopy (FSM).

يعيش التصوير خلية من طراز F - أكتين حيوية عن طريق الميكروسكوب الرقطة نيون (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

باستخدام نظام التقطير GELFREE 8100 لتقطير الوزن الجزيئي وبناء مع مرحلة الانتعاش السائل

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Linearization of the Bradford Protein Assay

Determination of microgram quantities of protein in the Bradford Coomassie brilliant blue assay is accomplished by measurement of absorbance at 590 nm. This most common assay enables rapid and simple protein quantification in cell lysates, cellular fractions, or recombinant protein samples, for the purpose of normalization of biochemical measurements. However, an intrinsic nonlinearity compromises the sensitivity and accuracy of this method. It is shown that under standard assay conditions, the ratio of the absorbance measurements at 590 nm and 450 nm is strictly linear with protein concentration. This simple procedure increases the accuracy and improves the sensitivity of the assay about 10-fold, permitting quantification down to 50 ng of bovine serum albumin. Furthermore, the interference commonly introduced by detergents that are used to create the cell lysates is greatly reduced by the new protocol. A linear equation developed on the basis of mass action and Beer's law perfectly fits the experimental data.

The accuracy and sensitivity of protein determination by the rapid and convenient Bradford assay is compromised by intrinsic nonlinearity. We show a simple linearization procedure that greatly increases the accuracy, improves the sensitivity of the assay about 10-fold, and significantly reduces interference by detergents.

الخطية من الفحص البروتين برادفورد

Determination of microgram quantities of protein in the Bradford Coomassie brilliant blue assay is accomplished by measurement of absorbance at 590 nm. ...

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

PAR كليب -- وسيلة لتحديد نطاق Transcriptome مواقع الربط من البروتينات الحمض النووي الريبي تجليد

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

Study of the Actin Cytoskeleton in Live Endothelial Cells Expressing GFP-Actin

The microvascular endothelium plays an important role as a selectively permeable barrier to fluids and solutes. The adhesive junctions between endothelial cells regulate permeability of the endothelium, and many studies have indicated the important contribution of the actin cytoskeleton to determining junctional integrity1-5. A cortical actin belt is thought to be important for the maintenance of stable junctions1, 2, 4, 5. In contrast, actin stress fibers are thought to generate centripetal tension within endothelial cells that weakens junctions2-5. Much of this theory has been based on studies in which endothelial cells are treated with inflammatory mediators known to increase endothelial permeability, and then fixing the cells and labeling F-actin for microscopic observation. However, these studies provide a very limited understanding of the role of the actin cytoskeleton because images of fixed cells provide only snapshots in time with no information about the dynamics of actin structures5. 
Live-cell imaging allows incorporation of the dynamic nature of the actin cytoskeleton into the studies of the mechanisms determining endothelial barrier integrity. A major advantage of this method is that the impact of various inflammatory stimuli on actin structures in endothelial cells can be assessed in the same set of living cells before and after treatment, removing potential bias that may occur when observing fixed specimens. Human umbilical vein endothelial cells (HUVEC) are transfected with a GFP-&beta;-actin plasmid and grown to confluence on glass coverslips. Time-lapse images of GFP-actin in confluent HUVEC are captured before and after the addition of inflammatory mediators that elicit time-dependent changes in endothelial barrier integrity. These studies enable visual observation of the fluid sequence of changes in the actin cytoskeleton that contribute to endothelial barrier disruption and restoration. 
Our results consistently show local, actin-rich lamellipodia formation and turnover in endothelial cells. The formation and movement of actin stress fibers can also be observed. An analysis of the frequency of formation and turnover of the local lamellipodia, before and after treatment with inflammatory stimuli can be documented by kymograph analyses. These studies provide important information on the dynamic nature of the actin cytoskeleton in endothelial cells that can used to discover previously unidentified molecular mechanisms important for the maintenance of endothelial barrier integrity.

Microscopic imaging of live endothelial cells expressing GFP-actin allows characterization of dynamic changes in cytoskeletal structures. Unlike techniques that use fixed specimens, this method provides a detailed assessment of temporal changes in the actin cytoskeleton in the same cells before, during, and after various physical, pharmacological, or inflammatory stimuli.

دراسة الهيكل الخلوي أكتين في الخلايا البطانية لايف تعرب عن GFP - أكتين

The microvascular endothelium plays an important role as a selectively permeable barrier to fluids and solutes. The adhesive junctions between endothelial ...

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.

تقارب المرحلة السائلة الفحص التقاطها باستخدام الخرز المغناطيسي لدراسة البروتين البروتين التفاعل: المثال فيروس شلل الأطفال، Nanobody

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

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

A تجليد الفحص إنتاجية عالية MHC II لتحليل الكمي لالببتيد الحواتم

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

Reporter-based Growth Assay for Systematic Analysis of Protein Degradation

Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard approach to determining intracellular protein degradation relies on biochemical assays for following the kinetics of protein decline. Such methods are often laborious and time consuming and therefore not amenable to experiments aimed at assessing multiple substrates and degradation conditions. As an alternative, cell growth-based assays have been developed, that are, in their conventional format, end-point assays that cannot quantitatively determine relative changes in protein levels.
Here we describe a method that faithfully determines changes in protein degradation rates by coupling them to yeast cell-growth kinetics. The method is based on an established selection system where uracil auxotrophy of URA3-deleted yeast cells is rescued by an exogenously expressed reporter protein, comprised of a fusion between the essential URA3 gene and a degradation determinant (degron). The reporter protein is designed so that its synthesis rate is constant whilst its degradation rate is determined by the degron. As cell growth in uracil-deficient medium is proportional to the relative levels of Ura3, growth kinetics are entirely dependent on the reporter protein degradation.
This method accurately measures changes in intracellular protein degradation kinetics. It was applied to: (a) Assessing the relative contribution of known ubiquitin-conjugating factors to proteolysis (b) E2 conjugating enzyme structure-function analyses (c) Identification and characterization of novel degrons. Application of the degron-URA3-based system transcends the protein degradation field, as it can also be adapted to monitoring changes of protein levels associated with functions of other cellular pathways.

Here we describe a robust biological assay for quantifying the relative rate of proteolysis by the ubiquitin-proteasome system. The assay readout is yeast growth rate in liquid culture, which is dependent on the cellular levels of a reporter protein comprising a degradation signal fused to an essential metabolic marker.

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Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard ...