Bu kağıt, burada açıklanan orijinal araştırma ev sahipliği yapan merhum Dr. Zvi Selinger anısına adanmış.

Bradford Protein Kalibrasyon Grafik Doğrusallaştırma: 1 Bradford testinin yaygın olarak bilinen Coomassie parlak mavi protein tahlil, yaygın, hızlı ve kullanışlı bir protokol yanı sıra bağıl duyarlılık nedeniyle kullanılır . Ne yazık ki, büyük ölçüde protein konsantrasyonları (Şekil 1) üzerinde geniş bir eğrilik var. Bu nedenle, nispeten yüksek protein konsantrasyonları, 2-10 mg / ml BSA, sadece dar bir aralıkta daha iyi (Şekil 1, yeşil) lineer regresyon uygun kalibrasyon grafiği için kullanılır. Ancak, doğrusal olmayan büyük bir hata önlemek için kalibrasyon grafiği sınırlı giren bilinmeyen örneklerin protein konsantrasyonu gerektirir ve aynı zamanda sınırlı aralığında hassasiyeti azaltır. Doğrusal mikrogram miktarda protein mevcut değildir, özellikle ciddi bir sorun sunar ve genellikle bilinmeyen örneklerin birden fazla dilüsyonları gerektirir. Orijinal Bradford yazıda belirtildiği gibi, &quot;doğrusal kaynak, boya iki farklı renk formları spektrumunun bir çakışma olmadığından reaktif kendisidir.&quot; 1. Aslında, üç tür Coomassie parlak mavi boya 2 testin normal asidik pH, asit-baz dengesi . Kırmızı, mavi ve yeşil formları sırasıyla 590, 470 ve 650 nm (Şekil 2) absorbans maxima var. Mavi, yoğun, 594 nm 3, 4 (Şekil 2) ışık emer karmaşık bir şekillendirme, protein bağlayan şeklidir. Bradford 1 (Şekil 3) &quot;reaktif için arka plan değeri daha fazla boya proteine ​​bağlı olarak sürekli azalmaktadır&quot; diye kaydetti. Bu nedenle, artan protein miktarları eklenir, protein boyası kompleksi emmez, 450 nm dalga boyunda absorbans değişimi ölçerek, 590 nm arka plan azalma hesaplamak için çalıştı. Bulduğumuz azalan arka plan kısmen değil, tamamen doğrusal hesapları (Şekil 4). Protein-boya bağlayıcı bir denge 5 olduğu gibi, çünkü, sonra ücretsiz boya konsantrasyonunda azalma doğrusal yanıt başka bir bozulma ürettiği hipotezi, kompleks oluşum değil, aynı zamanda ücretsiz boya, sadece protein konsantrasyonu bağlıdır. (Şekil 5). Değişken ücretsiz boya konsantrasyonu ile ilgili hem konular göz önüne alırsak, biz, protein konsantrasyon ve absorbans ölçümleri 590 nm, 450 nm (Şekil 6) üzerinde oranı arasında doğrusal bir ilişki tanımlayan matematiksel bir denklem geliştirdi. Teorik ve deneysel çalışmanın ayrıntılı bir açıklama Analitik Biyokimya 6 1996 yayında bulunabilir. Matematiksel denklemi deneysel olarak test edilmiş ve tüm protein konsantrasyonları aralığı (Şekil 7) üzerinde lineer bir kalibrasyon eğrisi verimi tespit edildi. Ayrıca, denklem Y ekseni önünü 6, doğru pH-bağımlı değeri bağımsız bir kararlılık tarafından da doğrulandı . Geliştirilmiş Bradford Protein Testi için ayrıntılı Protokolü, Mikroplaka Absorbans Okuyucu: <ol><li> Standart, sığır serum albumin 0.1 mg / ml stok solüsyonu hazırlayın. Herhangi bir diğer standart seçilmiş, ancak aynı standart tüm deneylerde kullanılan gerektiğini unutmayın olabilir. </li><li> Bilinmeyen örnekler deiyonize su ile seyreltilir. 5-50 mg / ml hedefliyoruz. Ancak, testin doğrusal aralığı için belirgin bir sınır olmadığı için daha yüksek veya daha düşük protein konsantrasyonları, kabul edilebilir. Ancak, ölçüm absorbans okuyucu doğrusal aralığı içinde olmalıdır. </li><li> Deiyonize su Bradford reaktifi (Bio-Rad) 2.5 kat sulandırınız. </li><li> 0 ve üç nüsha kuyuları (0-5 mg BSA kalibrasyon eğrisi oluşturma) BSA stok solüsyonu 10-50 ul ekleyin. / Ul 100 ulaşmak için deiyonize su ile tamamlayın. </li><li> Farklı bir kuyu, 100 ul triplicates bilinmeyen örnek ekleyin. Bilinmeyen örnek çeşitli konsantrasyonlarda doğruluğunu artırmak için kullanılıyor olabilir. </li><li> Tüm kuyulara seyreltilmiş Bradford reaktifi 100 ul ekleyin. Toplam hacmi 200 ul / iyi. </li><li> En az 5 dakika değil, renk gelişimi için fazla 60 dakika bekleyin. </li><li> Boş deiyonize su (ve sıfır protein boyası) 200 ul olmalıdır. </li><li> 590 nm ve 450 nm dalga boyunda absorbansı ölçülür. </li><li> , 590 nm net absorbans değerleri bölerek ve 450 nm dalga boyunda bir kalibrasyon grafiği hazırlayın. Sıfır protein (sadece boya) değer bir veri noktası (Şekil 8) olarak dahil olması gerektiğini unutmayın. </li><li> Kalibrasyon eğrisi (Şekil 9) lineer denklem dayalı bilinmeyen numune konsantrasyonu hesaplayın. </li></ol> Temsilcisi Sonuçlar: Absorbans 590 nm tek bir dalga boyu, absorbans değerleri, 590 nm 450 nm üstü oranı aksine, lineer wi. protein konsantrasyonu (Şekil 10). Bilinmeyen örnek protein konsantrasyonu kalibrasyon eğrisi (Şekil 10, denklem) lineer denklem kullanılarak hesaplanmıştır olabilir. Ancak, artan doğruluğu bilinmeyen birkaç numune dilüsyonları ölçerek elde edilir. Bu amaçla, iki grafikler hazırlar. Ilk X ekseni (Şekil 11, sağ) mg protein ile standart bir kalibrasyon grafiği. İkinci grafikte X ekseni (Şekil 11, sol) orijinal sulandırılmamış örnek ul, bilinmeyen bir örnek. Boya sadece değeri (Şekil 11) her iki grafiklerde dahil edilmelidir. Bilinmeyen örnek protein konsantrasyonu bilinmeyen bir örnek ve standart eteklerinde (Şekil 11, denklem) bölünmesi ile elde edilir. <img src="/files/ftp_upload/1918/1918fig2.jpg" alt="Şekil 1" /> Şekil 1. Konvansiyonel Bradford kalibrasyon grafiği. Doğrusal aralığı yeşil sembollerle temsil edilmektedir. <img src="/files/ftp_upload/1918/1918fig1.jpg" alt="Şekil 2" /> Şekil 2 protein-boya kompleks ve yalnız boya Spectra. <img src="/files/ftp_upload/1918/1918fig3.jpg" alt="Şekil 3" /> Şekil 3. Protein-boya denge. <img src="/files/ftp_upload/1918/1918fig4.jpg" alt="Şekil 4" /> Şekil 4 doğru hesaplanan arka plan Azaltma kısmen doğrusal azalır. <img src="/files/ftp_upload/1918/1918fig5.jpg" alt="Şekil 5" /> Şekil 5 Protein-boya denge. <img src="/files/ftp_upload/1918/1918fig6.jpg" alt="Şekil 6" /> Şekil 6 Bradford protein testinin doğrusallaştırma için matematiksel olarak. <img src="/files/ftp_upload/1918/1918fig7.jpg" alt="Şekil 7" /> Şekil 7 Bradford kalibrasyon grafiği Doğrusallaştırma. <img src="/files/ftp_upload/1918/1918fig8.jpg" alt="Şekil 8" /> Şekil 8 absorbans oranı değerlerinin hesaplanması. <img src="/files/ftp_upload/1918/1918fig9.jpg" alt="Şekil 9" /> Şekil 9 A lineer Bradford kalibrasyon grafiği. <img src="/files/ftp_upload/1918/1918fig10.jpg" alt="Şekil 10" /> Şekil 10 Bradford kalibrasyon grafiği Doğrusallaştırma. <img src="/files/ftp_upload/1918/1918fig11.jpg" alt="Şekil 11" /> Şekil 11 Bilinmeyen numune konsantrasyonu hesaplama.

<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>

Bradford protein tahlil performans ve bağıl duyarlılık kolaylığı nedeniyle popüler. Protokol ile elde edilen aralığı tüm protein konsantrasyonları üzerine doğrusallaştırma, bilinmeyen örnekler aralığında kalibrasyon grafiği düşmeye gerek yok, burada daha fazla testin kolaylaştırır sundu. Önemlisi, daha da geliştirilmiş protokol orijinal Bradford protokolü üzerinden aşağıdaki avantajları sağlar: <ol><li> Doğruluğunu arttırdı. </li><li> Hassasiyet, ...

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<td>Bradford reagent</td>
<td>&nbsp;</td>
<td>Bio-Rad Laboratories</td>
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<td>Bovine Serum Albumin</td>
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linearization of the bradford protein assay

Bradford Coomassie parlak mavi tayininde protein mikrogram miktarların belirlenmesi, 590 nm&#39;de absorbans ölçümü ile gerçekleştirilir. Bu en yaygın test biyokimyasal ölçümleri normalleştirme amacıyla, hızlı ve basit bir protein, hücre lizatları, hücresel kesirler, ya da rekombinant protein örneklerinin kantifikasyon sağlar. Ancak, içsel bir doğrusal bu yöntemin duyarlılığı ve hassasiyeti uzlaşır. Bu, standart test koşulları altında 590 nm&#39;de absorbans ölçümleri ve 450 nm dalga oranı, protein konsantrasyonu ile kesinlikle doğrusal olduğunu göstermiştir. Bu basit bir prosedür doğruluğunu artırır ve 50 ng bovine serum albumin, 10 kat izin kantifikasyon hakkında testinin duyarlılığını artırır. Ayrıca, sık hücre lizatları oluşturmak için kullanılan deterjanların piyasaya girişim yeni bir protokol tarafından büyük ölçüde azalır. Kitlesel eylem ve Beer yasası temelinde geliştirilen bir lineer denklem deneysel veriler mükemmel uyuyor.

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

Linearization of the Bradford Protein Assay

Hızlı ve rahat Bradford yöntemi ile protein tayini doğruluk ve hassasiyet içsel doğrusal tehlikeye düşer. Biz büyük ölçüde hassasiyeti artırır yaklaşık 10 kat testin duyarlılığını artırır ve anlamlı deterjanların müdahaleleri azaltıyor, basit bir doğrusallaştırma prosedürü göstermektedir.

Bradford Protein Testi lineerleştirilmesi

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

Research

JoVE Journal

Biology

98.3K Görüntüleme.  Tel Aviv University. Hızlı ve rahat Bradford yöntemi ile protein tayini doğruluk ve hassasiyet içsel doğrusal tehlikeye düşer. Biz büyük ölçüde hassasiyeti artırır yaklaşık 10 kat testin duyarlılığını artırır ve anlamlı deterjanların müdahaleleri azaltıyor, basit bir doğrusallaştırma prosedürü göstermektedir.

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.

Aktin Co-Sedimantasyon Testi; F-Aktin Bağlayıcı Protein Analizi

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

Biyoloji

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.

Protein Ubikitinasyon Tespiti

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.

Organik Solvent ve Asetik Asit olmadan Jeller Coomassie G-250 ile Proteinlerin Boyama

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.

Sıvı Faz Kurtarma Moleküler Ağırlık Tabanlı Fraksiyonu GELFREE 8100 Fraksiyonu Sisteminin Kullanılması

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)

Helios Gen Tabancası kullanarak Soğan Hücrelerde Protein-Protein Etkileşimi Bimoleküler Floresan tamamlama (BiFC) Testi

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.

Protein-Protein Etkileşimi Okumak Manyetik Boncuk Kullanarak Bir Sıvı Faz Affinity Yakalama Testi: Poliovirüs-Nanobody Örnek

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.

Predasyon Risk, Herbivore Fizyolojik Stres ve Bitki Çöp mikrobiyal bozunma Bağlama

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.

Protein Parçalanabilirliklerinin Sistematik Analizi muhabiri tabanlı Büyüme Deneyi

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.

Kantitatif İmmünofloresan Deneyi sentrozomlar Protein Düzeyleri Varyasyon Tedbir

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

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

RNA/protein interactions are critical for post-transcriptional regulatory pathways. Among the best-characterized cytosolic RNA-binding proteins are iron regulatory proteins, IRP1 and IRP2. They bind to iron responsive elements (IREs) within the untranslated regions (UTRs) of several target mRNAs, thereby controlling the mRNAs translation or stability. IRE/IRP interactions have been widely studied by EMSA. Here, we describe the EMSA protocol for analyzing the IRE-binding activity of IRP1 and IRP2, which can be generalized to assess the activity of other RNA-binding proteins as well. A crude protein lysate containing an RNA-binding protein, or a purified preparation of this protein, is incubated with an excess of32 P-labeled RNA probe, allowing for complex formation. Heparin is added to preclude non-specific protein to probe binding. Subsequently, the mixture is analyzed by non-denaturing electrophoresis on a polyacrylamide gel. The free probe migrates fast, while the RNA/protein complex exhibits retarded mobility; hence, the procedure is also called &#8220;gel retardation&#8221; or &#8220;bandshift&#8221; assay. After completion of the electrophoresis, the gel is dried and RNA/protein complexes, as well as free probe, are detected by autoradiography. The overall goal of the protocol is to detect and quantify IRE/IRP and other RNA/protein interactions. Moreover, EMSA can also be used to determine specificity, binding affinity, and stoichiometry of the RNA/protein interaction under investigation.

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

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

IRE / IRP Örnek: RNA-protein Etkileşimleri Çalışmaları elektroforetik hareketlilik Shift Testi (EMSA)

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