本論文では、ここで説明する独自の研究をホストする故博士Zvi Selinger、の記憶に捧げられています。

Bradfordタンパク質のキャリブレーションのグラフの線形化： 一般的にブラッドフォードアッセイ1として知られているクマシーブリリアントブルータンパク質アッセイは、広くために迅速かつ便利なプロトコルだけでなく、その相対的な感度で使用されています。残念なことに、タンパク質濃度の広い範囲（図1）上の曲率の大きい程度がある。そのため、比較的高いタンパク質濃度の唯一の狭い範囲、20〜10 mg / mlのBSAは、次により良い線形回帰（図1、緑）に適合キャリブレーショングラフ、のために使用されます。しかし、非線形性が大きな誤差を避けるためにキャリブレーションのグラフの限られた範囲内に未知のサンプルのタンパク質濃度を必要とし、それも限られた範囲内で精度が低下します。非線形性は、タンパク質のマイクログラムの量が利用できない、特に深刻な問題を提示し、それは多くの場合、未知のサンプルの複数の希釈を必要とします。 オリジナルのブラッドフォードの論文で述べたように、&quot;非線形性の源は、染料の2つの異なる色形のスペクトルに重なりがあるので。試薬自体にある&quot;1。実際には、クマシーブリリアントブルー色素の3つの形式は、アッセイ2の通常の酸性pHでの酸塩基平衡状態にある。赤、青、緑のフォームは、470でそれぞれ590と650nm、（図2）吸光度の最大値を持っている。青は激しく594 nmの3、4（図2）で光を吸収する複合体を形成、タンパク質を結合するフォームです。ブラッドフォードはまた、1（図​​3）&quot;より多くの色素がタンパク質に結合されている試薬のバックグラウンド値が継続的に減少している&quot;と指摘した。したがって、我々はタンパク質の量を増やすとして590nmでの背景の削減を計算しようとしたタンパク質 - 色素複合体が吸収しない450nmの、吸光度の変化を測定することによって、追加されます。我々は、部分的にその減少の背景を発見ではなく、完全に、非線形性のためのアカウント（図4）。 我々は、その後、タンパク質-色素結合が平衡5にあるように、複合体形成だけではなく、自由なタンパク質の濃度だけでなく、無料の染料のそれに依存するため、遊離色素濃度の減少は、線形応答の別の歪みを生成するという仮説を立てた（図5）。考慮フリー染料の変数濃度に関連した両方の問題を踏まえ、我々はタンパク質濃度と吸光度測定、450 nmの（図6）以上590nmの比の間に直線関係を記述する数学的方程式を開発した。理論的および実験的研究の詳細な説明は、アナリティカルバイオケミストリー（Analytical Biochemistry）6の私たちの1996年出版で見つけることができます。数学的な方程式は、実験的にテストされ、全体のタンパク質濃度の範囲（図7）で直線の検量線が得られることがわかった。また、式はY軸切片6の正しいpH依存性値の独立した決定によっても検証されました。 改良されたブラッドフォードプロテインアッセイのための詳細なプロトコル、マイクロプレート吸光度リーダーを使用して： <ol><li>標準、ウシ血清アルブミン0.1 mg / mlのストック溶液を調製。他の標準を選択しますが、同じ規格が全ての実験で使用する必要があることに注意してくださいすることができます。 </li><li>脱イオン水で未知サンプルを希釈する。 5-50μg/ mlのを目指しています。アッセイの直線範囲のための明白な限界がないのでまだ、高いか低いタンパク質濃度は、許容範囲です。しかし、測定は、吸光度リーダーの直線範囲内でなければなりません。 </li><li>ブラッドフォード試薬（Bio - Rad社製）2.5倍の脱イオン水で希釈してください。 </li><li> 3つのウェルにBSAの原液（0-5μgのBSAの検量線を作成する）の0および10〜50μlを添加します。 100μl/ウェルを達するために脱イオン水で補う。 </li><li>別の井戸では、三連で未知のサンプルの100μlを加える。未知のサンプルのいくつかの濃度は、精度を高めるために使用されることがあります。 </li><li>すべてのウェルに希釈したBradford試薬100μLを加える。総ボリュームは十分に200μl/です。 </li><li>少なくとも5分ではなく、発色のための60以上の分を待ちます。 </li><li>ブランクは、脱イオン水（とではないだけでなく、ゼロタンパク質の色素）を200μlにする必要があります 。 </li><li> 590nmで450nmの吸光度を測定します。 </li><li> 590nmで正味吸光度値を割って、450 nmでのキャリブレーショングラフを準備します。ゼロタンパク質（染料のみ）の値をデータ点（図8）として含まれるべきであることに注意してください。 </li><li>検量線（図9）の線形方程式に基づいて、未知サンプルの濃度を計算する。 </li></ol>代表的な結果： 590nmの吸光度値の比は、450 nmの上に590nmの単一波長で吸光度とは異なり、線形wはi番目のタンパク質濃度（図10）。 未知試料のタンパク質濃度は、単に検量線（図10、方程式）の線形方程式を用いて計算することができます。 しかし、精度を高めるには、いくつかの未知のサンプルの希釈液を測定して得られる。この目的のために、二つのグラフを用意。最初はX軸（図11、右）上μgのタンパク質を持つ標準のためのキャリブレーショングラフです。二つ目のグラフはX軸（図11、左）の元の未希釈サンプルの液で、未知のサンプル用です。染料のみの値は両方のグラフ（図11）に含まれるべきです。未知試料のタンパク質濃度は、未知試料と標準（図11、方程式）の斜面を分割することによって導出されます。 <img src="/files/ftp_upload/1918/1918fig2.jpg" alt="図1" /> 図1従来のブラッドフォードのキャリブレーショングラフ。直線性の範囲は、緑の記号で表されます。 <img src="/files/ftp_upload/1918/1918fig1.jpg" alt="図2" /> 図2。タンパク質-色素複合体の単独で色素のスペクトル。 <img src="/files/ftp_upload/1918/1918fig3.jpg" alt="図3" /> 図3。タンパク質-色素平衡。 <img src="/files/ftp_upload/1918/1918fig4.jpg" alt="図4" /> 図4。正しい計算されたバックグラウンドの低減は、部分的に非線形性を減少させる。 <img src="/files/ftp_upload/1918/1918fig5.jpg" alt="図5" /> 図5。タンパク質-色素平衡。 <img src="/files/ftp_upload/1918/1918fig6.jpg" alt="図6" /> 図6。ブラッドフォードタンパク質アッセイの線形化のための数学的基礎。 <img src="/files/ftp_upload/1918/1918fig7.jpg" alt="図7" /> 図7。ブラッドフォードのキャリブレーションのグラフの線形化。 <img src="/files/ftp_upload/1918/1918fig8.jpg" alt="図8" /> 図8。吸光度比の値の計算。 <img src="/files/ftp_upload/1918/1918fig9.jpg" alt="図9" /> 図9。線形ブラッドフォードのキャリブレーショングラフ。 <img src="/files/ftp_upload/1918/1918fig10.jpg" alt="図10" /> 図10ブラッドフォードのキャリブレーションのグラフの線形化。 <img src="/files/ftp_upload/1918/1918fig11.jpg" alt="図11" /> 図11。未知のサンプル濃度の計算。

<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>

ブラッドフォードタンパク質アッセイは、原因で、パフォーマンスと相対感度のその使いやすさに人気があります。プロトコルによって得られた距離、タンパク質全体の濃度以上の線形化は、未知のサンプルは、キャリブレーションのグラフの範囲内にする必要がないようにここでさらに、アッセイを簡素化発表。 重要なのは、改良されたプロトコルでは、さらにオリ?...

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<td>Bradford reagent</td>
<td>&nbsp;</td>
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<td>Bovine Serum Albumin</td>
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<td>Amersco</td>
<td>0332</td>
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linearization of the bradford protein assay

ブラッドフォードクマシーブリリアントブルーアッセイにおけるタンパク質のマイクログラム量の決定は、590nmでの吸光度を測定することによって達成される。この最も一般的なアッセイは、生化学的測定値の正規化の目的のために、細胞ライセート、細胞の分画、または組換えタンパク質サンプルの迅速でシンプルなタンパク質の定量が可能になります。しかし、本質的な非線形性は、この方法の感度と精度が低下します。それは標準的なアッセイ条件下で、590 nmと450 nmの吸光度測定の比はタンパク質濃度と厳密に線形であることが示されている。このシンプルな手順では、精度を向上させ、ダウンウシ血清アルブミンの50ngの10倍、許可定量化に関するアッセイの感度を向上させます。さらに、一般的に細胞ライセートを作成するために使用される界面活性剤によって導入された干渉が大幅に新しいプロトコルによって低減されます。線形方程式は、質量作用に基づいて開発され、ベールの法則は完璧に実験データをフィット。

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

Linearization of the Bradford Protein Assay

迅速かつ便利なブラッドフォードアッセイによるタンパク質定量の精度と感度は本質的な非線形性によって危険にさらされます。我々は、大幅に精度を向上させる簡単な線形化法を示して10倍程度アッセイの感度が向上し、大幅な洗剤による干渉を低減します。

ブラッドフォードプロテインアッセイの線形化

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

97.9K ビュー.  Tel Aviv University. 迅速かつ便利なブラッドフォードアッセイによるタンパク質定量の精度と感度は本質的な非線形性によって危険にさらされます。我々は、大幅に精度を向上させる簡単な線形化法を示して10倍程度アッセイの感度が向上し、大幅な洗剤による干渉を低減します。

ビデオ: 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.

アクチン共沈降アッセイ、F -アクチンへの結合タンパク質の解析のための

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

生物学

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.

タンパク質のユビキチン化の検出

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.

クーマシー有機溶媒および酢酸のないG - 250でゲル中のタンパク質の染色

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.

液相の回復と分子量ベース画用GELFREE 8100分別システムの使用

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)

ヘリオス遺伝子銃を用いたタマネギの細胞における蛋白質 - 蛋白質相互作用のための二分子蛍光相補（BiFC）アッセイ

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.

タンパク質 - タンパク質相互作用を研究するために磁気ビーズを用いて液相アフィニティキャプチャーアッセイ：ポリオ-nanobodyの例

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.

捕食リスク、草食動物生理的ストレスと植物リターの微生物分解を結ぶ

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.

タンパク質分解の体系的な分析のためのレポーターベースの成長アッセイ

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.

中心体におけるタンパク質レベルの変化を測定するための定量的免疫蛍光アッセイ

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例：RNA  - タンパク質相互作用の研究のための電気泳動移動度シフトアッセイ（EMSA）

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