Questo saggio è dedicato alla memoria del compianto Dr. Zvi Selinger, che ha ospitato la ricerca originale descritta qui.

Linearizzazione della curva di taratura Proteine ​​Bradford: Il brillante saggio di proteine ​​Coomassie blu, comunemente noto come il saggio Bradford 1, è ampiamente utilizzato per il suo protocollo rapidi e convenienti così come la sua sensibilità relativa. Purtroppo, c&#39;è un ampio grado di curvatura su una vasta gamma di concentrazioni di proteine ​​(Fig. 1). Pertanto, solo una gamma ristretta di relativamente alte concentrazioni di proteine, 2-10 mg / ml BSA, viene utilizzato per la curva di taratura, che poi si adatta meglio di regressione lineare (Fig. 1, verde). Tuttavia, la non linearità richiede una concentrazione proteica dei campioni sconosciuti rientrano nella gamma limitata di curva di taratura allo scopo di evitare un errore grande, e riduce anche la precisione nel range limitato. La non linearità presenta un serio problema in particolare quando quantità microgrammi di proteina non sono disponibili, e richiede spesso più diluizioni dei campioni sconosciuti. Come sottolineato nel documento originale Bradford, &quot;la fonte della non linearità è nel reagente stesso poiché non vi è una sovrapposizione nello spettro delle due forme colore diverso del colorante.&quot; 1. Infatti, tre forme del colorante Coomassie blu brillante in equilibrio acido-base al pH acido del solito saggio 2. Le forme rosso, blu e verde sono assorbanza massimi a 470, 590 e 650 nm, rispettivamente (Fig. 2). Il blu è la forma che lega la proteina, formando un complesso che assorbe intensamente la luce a 594 nm 3, 4 (Fig. 2). Bradford ha anche osservato che &quot;il valore di fondo per il reagente è in costante riduzione come più colorante si lega alle proteine&quot; 1 (Fig. 3). Pertanto, abbiamo cercato di calcolare la riduzione dello sfondo 590 nm come quantità proteico sono aggiunti, misurando la variazione di assorbanza a 450 nm, in cui la proteina-colorante non assorbe. Abbiamo trovato che lo sfondo diminuendo in parte, ma non completamente, spiega la non-linearità (Fig. 4). Abbiamo quindi ipotizzato che la diminuzione della concentrazione di colorante libero produce un&#39;altra distorsione della risposta lineare, perché, come proteina-colorante legame è in equilibrio 5, formazione di complessi dipende non solo dalla concentrazione della proteina libero, ma anche su quello del colorante libero (Fig. 5). Tenendo conto sia questioni legate alla concentrazione variabile del colorante libero, abbiamo sviluppato una equazione matematica che descrive una relazione lineare tra la concentrazione di proteine ​​e il rapporto delle misure di assorbanza, 590 nm oltre 450 nm (Fig. 6). Una descrizione dettagliata dello studio teorico e sperimentale si possono trovare nel nostro 1996 pubblicazione nella biochimica analitica 6. L&#39;equazione matematica è stata sperimentalmente testato ed è risultato fino ad ottenere una curva di calibrazione lineare su tutta la gamma concentrazioni di proteine ​​(Fig. 7). Inoltre, l&#39;equazione è stata convalidata anche da una determinazione indipendente dal corretto pH-dipendente valore della Y intercettare 6. Protocollo dettagliato per la migliore analisi delle proteine ​​Bradford, utilizzando un lettore di micropiastre Assorbanza: <ol><li> Preparare un 0,1 mg / ml soluzione madre dello standard, albumina sierica bovina. Tutte le norme possono essere scelti altri, ma si noti che lo stesso standard deve essere usato in tutti gli esperimenti. </li><li> Diluire i campioni sconosciuti in acqua deionizzata. Puntate a 5-50 mg / ml. Tuttavia, le concentrazioni di proteine ​​superiori o inferiori sono accettabili, poiché non vi è alcun limite apparente per la gamma lineare del test. Tuttavia, la misura deve essere all&#39;interno della gamma lineare del lettore assorbanza. </li><li> Diluire il reagente di Bradford (Bio-Rad) 2,5 volte in acqua deionizzata. </li><li> Aggiungi 0 e 10-50 ml di soluzione madre di BSA pozzi triplice copia (la creazione di una curva di calibrazione mcg 0-5 BSA). Complemento con acqua deionizzata per raggiungere 100 l / pozzetto. </li><li> In diversi pozzetti, aggiungere 100 ml di campione sconosciuto in triplicato. Diverse concentrazioni del campione sconosciuto può essere utilizzato per aumentare la precisione. </li><li> Aggiungere 100 l di reagente diluito Bradford a tutti i pozzetti. Volume totale è di 200 microlitri / bene. </li><li> Attendere almeno 5 minuti, ma non più di 60 minuti per lo sviluppo del colore. </li><li> Il vuoto deve essere di 200 ml di acqua deionizzata (e non il colorante proteico pari a zero bene). </li><li> Misurare l&#39;assorbanza a 590 nm e 450 nm. </li><li> Preparare una curva di taratura dividendo i valori di assorbanza netta a 590 nm e 450 nm. Si noti che la proteina zero (colorante solo) valore deve essere inclusa come un punto di dati (Fig. 8). </li><li> Calcolare la concentrazione del campione sconosciuto in base all&#39;equazione lineare della curva di calibrazione (Fig. 9). </li></ol> Rappresentante dei risultati: A differenza l&#39;assorbanza ad una singola lunghezza d&#39;onda di 590 nm, il rapporto tra i valori di assorbanza, più di 590 nm a 450 nm, è lineare wconcentrazione di proteine-esimo (fig. 10). La concentrazione proteica del campione sconosciuto può essere semplicemente calcolato utilizzando l&#39;equazione lineare della curva di calibrazione (Fig. 10, l&#39;equazione). Tuttavia, una maggiore precisione si ottiene misurando diverse diluizioni del campione sconosciuto. A tal fine, preparare due grafici. La prima è una curva di taratura per lo standard con proteine ​​mcg sull&#39;asse X (Fig. 11, a destra). Il secondo grafico è per il campione incognito, con l di campione originale non diluito sull&#39;asse X (Fig. 11, a sinistra). Il valore di colorante solo dovrebbero essere incluse in entrambi i grafici (Fig. 11). La concentrazione proteica del campione sconosciuto si ottiene dividendo le pendici del campione sconosciuto e lo standard (Fig. 11, l&#39;equazione). <img src="/files/ftp_upload/1918/1918fig2.jpg" alt="Figura 1" /> Figura 1. Convenzionale grafico di calibrazione Bradford. Il range lineare è rappresentata da simboli verdi. <img src="/files/ftp_upload/1918/1918fig1.jpg" alt="Figura 2" /> Figura 2. Spectra della proteina-colorante e del colorante da solo. <img src="/files/ftp_upload/1918/1918fig3.jpg" alt="Figura 3" /> Figura 3. Proteina-colorante equilibrio. <img src="/files/ftp_upload/1918/1918fig4.jpg" alt="Figura 4" /> Figura 4. Riduzione del fondo corretta calcolato diminuisce parzialmente non-linearità. <img src="/files/ftp_upload/1918/1918fig5.jpg" alt="Figura 5" /> Figura 5. Proteina-colorante equilibrio. <img src="/files/ftp_upload/1918/1918fig6.jpg" alt="Figura 6" /> Figura 6. Fondamento matematico per la linearizzazione del dosaggio proteina Bradford. <img src="/files/ftp_upload/1918/1918fig7.jpg" alt="Figura 7" /> Figura 7. Linearizzazione della curva di taratura Bradford. <img src="/files/ftp_upload/1918/1918fig8.jpg" alt="Figura 8" /> Figura 8. Calcolo dei valori di rapporto tra l&#39;assorbanza. <img src="/files/ftp_upload/1918/1918fig9.jpg" alt="Figura 9" /> Figura 9. Una calibrazione lineare Bradford grafico. <img src="/files/ftp_upload/1918/1918fig10.jpg" alt="Figura 10" /> Figura 10. Linearizzazione della curva di taratura Bradford. <img src="/files/ftp_upload/1918/1918fig11.jpg" alt="Figura 11" /> Figura 11. Sconosciuto campione concentrazione calcolo.

<ol>
	<li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Anal Biochem. 72, 248-254 (1976).</li><li>Chial, H. J., Thompson, H. B., Splittgerber, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+spectral+study+of+the+charge+forms+of+Coomassie+blue+G.">A spectral study of the charge forms of Coomassie blue G.</a> Anal Biochem. 209, 258-266 (1993).</li><li>Chial, H. J., Splittgerber, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+the+binding+of+Coomassie+brilliant+blue+to+proteins+at+low+and+neutral+pH.">A comparison of the binding of Coomassie brilliant blue to proteins at low and neutral pH.</a> Anal Biochem. 213, 362-369 (1993).</li><li>Compton, S. J., Jones, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+dye+response+and+interference+in+the+Bradford+protein+assay.">Mechanism of dye response and interference in the Bradford protein assay.</a> Anal Biochem. 151, 369-374 (1985).</li><li>Congdon, R. W., Muth, G. W., Splittgerber, A. 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+binding+interaction+of+Coomassie+blue+with+proteins.">The binding interaction of Coomassie blue with proteins.</a> Anal Biochem. 213, 407-413 (1993).</li><li>Zor, T., Selinger, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linearization+of+the+Bradford+protein+assay+increases+its+sensitivity:+theoretical+and+experimental+studies.">Linearization of the Bradford protein assay increases its sensitivity: theoretical and experimental studies.</a> Anal Biochem. 236, 302-308 (1996).</li></ol>

Il dosaggio di proteine ​​Bradford è popolare grazie alla sua facilità di prestazioni e di sensibilità relativa. La linearizzazione sulle concentrazioni proteina intera gamma ottenuto dal protocollo presentato qui semplifica ulteriormente l&#39;analisi, come campioni sconosciuti non hanno bisogno di rientrare nel campo della curva di calibrazione. È importante sottolineare che il protocollo migliorata Provvede inoltre i seguenti vantaggi rispetto l&#39;originale Bradford protocollo: <ol><li> Aumento della precisione. </li><li> La sensibilità è aumentata di circa un ordine di grandezza, il che rende possibile determinare un minimo di 50 ng BSA nel test micropiastra 6. </li><li> La sensibilità permette una migliore quantificazione delle proteine ​​in presenza di detergenti. Con l&#39;originale Bradford protocollo, l&#39;interferenza da detergenti in genere utilizzato per lisi cellulare che spesso sfociano in una diminuita risposta alle proteine. Il miglioramento della sensibilità di un ordine di grandezza permette di diluizione dei campioni fino ad un punto in cui viene eliminata l&#39;interferenza con detergenti. Si prega di notare che i campioni standard dovrebbe contenere la stessa composizione detergente e concentrazione, come nei campioni diluiti sconosciuti. </li></ol> I picchi di assorbimento della proteina-colorante e la forma libera colorante rosso sono a 594 e 470 nm, rispettivamente. Si raccomanda di misurare l&#39;assorbanza a 590-600 nm, per ottenere una sensibilità massima di cambiare la concentrazione di proteine, e 450-485 nm, per garantire la linearizzazione. A causa della considerazione ottico lunghezza del percorso, una maggiore precisione può essere ottenuto in un test di 1 ml cui vengono collocati in una cuvette spettrofotometro regolare, piuttosto che un lettore di micropiastre. In questo caso, scale-up dei volumi di 5 volte. E &#39;assolutamente obbligatorio che l&#39;acqua deionizzata verrà utilizzato come vuota. Una curva di calibrazione lineare non può essere ottenuto se lo zero proteina standard di colorante contenente campione è usato come vuota.

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linearization of the bradford protein assay

Determinazione delle quantità di microgrammi di proteina nel saggio Bradford brillante blu Coomassie si ottiene la misura di assorbanza a 590 nm. Questo test permette di quantificare più comuni proteine ​​rapido e semplice in lisati di cellule, frazioni cellulari, o campioni di proteine ​​ricombinanti, ai fini della normalizzazione delle misure biochimiche. Tuttavia, un non-linearità intrinseca compromette la sensibilità e la precisione di questo metodo. E &#39;dimostrato che in condizioni di test standard, il rapporto tra le misure di assorbanza a 590 nm e 450 nm è strettamente lineare con la concentrazione di proteine. Questa semplice procedura aumenta la precisione e migliora la sensibilità del dosaggio circa 10 volte, permettendo la quantificazione fino al 50 ng di sieroalbumina bovina. Inoltre, l&#39;interferenza introdotta da detergenti comunemente utilizzati per creare i lisati cellulari è notevolmente ridotta dal nuovo protocollo. Una equazione lineare sviluppato sulla base di azione di massa e la legge Beer si adatta perfettamente ai dati sperimentali.

Watch this Scientific Journal Video about Linearization of the Bradford Protein Assay at JoVE.com

Linearization of the Bradford Protein Assay

La precisione e la sensibilità di determinazione delle proteine ​​da parte del test rapido e conveniente Bradford è compromessa dalla non linearità intrinseca. Mostriamo una semplice procedura di linearizzazione che aumenta notevolmente la precisione, migliora la sensibilità del test di circa 10 volte, e riduce significativamente le interferenze da detergenti.

Linearizzazione del saggio proteine ​​Bradford

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

linearization-of-the-bradford-protein-assay

Nanyang Technological University

Research

JoVE Journal

Biology

98.6K Views.  Tel Aviv University. La precisione e la sensibilità di determinazione delle proteine ​​da parte del test rapido e conveniente Bradford è compromessa dalla non linearità intrinseca. Mostriamo una semplice procedura di linearizzazione che aumenta notevolmente la precisione, migliora la sensibilità del test di circa 10 volte, e riduce significativamente le interferenze da detergenti.

Video: Linearization of the Bradford Protein Assay

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

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

Actina Co-sedimentazione di analisi, per l&#39;Analisi del Binding Protein a F-Actina

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

Ricerca

Biologia

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.

Rilevamento di proteine ​​Ubiquitinazione

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

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

La colorazione delle proteine ​​in gel con Coomassie G-250, senza solventi organici acido acetico e

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

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.

Utilizzando il sistema GELFREE 8100 Frazionamento per peso molecolare basata su Frazionamento con recupero della fase liquida

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

Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular environment. The Bimolecular Fluorescence Complementation (BiFC) Assay1 allows researchers to visualize protein-protein interactions in living cells and has become an essential research tool. This assay is based on the facilitated association of two fragments of a fluorescent protein (GFP) that are each fused to a potential interacting protein partner. The interaction of the two protein partners would facilitate the association of the N-terminal and C-terminal fragment of GFP, leading to fluorescence. For plant researchers, onion epidermal cells are an ideal experimental system for conducting the BiFC assay because of the ease in obtaining and preparing onion tissues and the direct visualization of fluorescence with minimal background fluorescence. The Helios Gene Gun (BioRad) is commonly used for bombarding plasmid DNA into onion cells. We demonstrate the use of Helios Gene Gun to introduce plasmid constructs for two interacting Arabidopsis thaliana transcription factors, SEUSS (SEU) and LEUNIG HOMOLOG (LUH)2 and the visualization of their interactions mediated by BiFC in onion epidermal cells.

Bimolecular Fluorescence Complementation BiFC Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

This article illustrates how to properly use the BioRad Helios Gene Gun to introduce plasmid DNA into onion epidermal cells and how to test for protein-protein interactions in onion cells based on the principle of Bimolecular Fluorescence Complementation (BiFC)

Biomolecolare fluorescenza complementazione (BiFC) Assay per interazione proteina-proteina nelle cellule Cipolla Utilizzando la Helios Gun Gene

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular ...

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.

Un liquido fase di cattura test affinità utilizzando biglie magnetiche per studiare interazioni proteina-proteina: il poliovirus-Nanobody Esempio

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

Linking Predation Risk, Herbivore Physiological Stress and Microbial Decomposition of Plant Litter

The quantity and quality of detritus entering the soil determines the rate of decomposition by microbial communities as well as recycle rates of nitrogen (N) and carbon (C) sequestration1,2. Plant litter comprises the majority of detritus3, and so it is assumed that decomposition is only marginally influenced by biomass inputs from animals such as herbivores and carnivores4,5. However, carnivores may influence microbial decomposition of plant litter via a chain of interactions in which predation risk alters the physiology of their herbivore prey that in turn alters soil microbial functioning when the herbivore carcasses are decomposed6. A physiological stress response by herbivores to the risk of predation can change the C:N elemental composition of herbivore biomass7,8,9 because stress from predation risk increases herbivore basal energy demands that in nutrient-limited systems forces herbivores to shift their consumption from N-rich resources to support growth and reproduction to C-rich carbohydrate resources to support heightened metabolism6. Herbivores have limited ability to store excess nutrients, so stressed herbivores excrete N as they increase carbohydrate-C consumption7. Ultimately, prey stressed by predation risk increase their body C:N ratio7,10, making them poorer quality resources for the soil microbial pool likely due to lower availability of labile N for microbial enzyme production6. Thus, decomposition of carcasses of stressed herbivores has a priming effect on the functioning of microbial communities that decreases subsequent ability to of microbes to decompose plant litter6,10,11.
We present the methodology to evaluate linkages between predation risk and litter decomposition by soil microbes. We describe how to: induce stress in herbivores from predation risk; measure those stress responses, and measure the consequences on microbial decomposition. We use insights from a model grassland ecosystem comprising the hunting spider predator (Pisuarina mira), a dominant grasshopper herbivore (Melanoplus femurrubrum),and a variety of grass and forb plants9.

We present methods to evaluate how predation risk can alter the chemical quality of herbivore prey by inducing dietary changes to meet demands of heightened stress, and how the decomposition of carcasses from these stressed herbivores slows subsequent plant litter decomposition by soil microbes.

Collegamento rischio di predazione, stress Herbivore fisiologica e decomposizione microbica dei Rifiuti vegetali

The quantity and quality of detritus entering the soil  determines the rate of decomposition by microbial communities as well as  recycle rates of ...

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.

Saggio di crescita a base Reporter, per l&#39;analisi sistematica di proteine ​​degradazione

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

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to facilitate sensory functions in cells. The level of a protein may be regulated differently at centrosomes than at other .cellular locations, and the variation in the centrosomal level of several proteins at different points of the cell cycle appears to be crucial for the proper regulation of centriole assembly. We developed a quantitative fluorescence microscopy assay that measures relative changes in the level of a protein at centrosomes in fixed cells from different samples, such as at different phases of the cell cycle or after treatment with various reagents. The principle of this assay lies in measuring the background corrected fluorescent intensity corresponding to a protein at a small region, and normalize that measurement against the same for another protein that does not vary under the chosen experimental condition. Utilizing this assay in combination with BrdU pulse and chase strategy to study unperturbed cell cycles, we have quantitatively validated our recent observation that the centrosomal pool of VDAC3 is regulated at centrosomes during the cell cycle, likely by proteasome-mediated degradation specifically at centrosomes.

Here, a novel quantitative fluorescence assay is developed to measure changes in the level of a protein specifically at centrosomes by normalizing that protein&#8217;s fluorescence intensity to that of an appropriate internal standard.

Quantitative Immunofluorescence Assay misurare la variazione dei livelli di proteine ​​ad centrosomi

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to ...

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein. Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein.
In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins.

Peptide array screening is a high throughput assay for identifying protein-protein interaction sites. This allows mapping multiple interactions of a target protein and can serve as a method for identifying sites for inhibitors that target a protein. Here we describe a protocol for screening and analyzing peptide arrays.

Identificare proteina-proteina siti di interazione Uso Peptide Array

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may ...

Linearizzazione del saggio proteine ​​Bradford

Linearizzazione del saggio proteine Bradford