本文是献给已故的博士兹维Selinger，谁主办的原创性研究，这里描述的内存。

Bradford蛋白校正曲线的线性化： 考马斯亮蓝蛋白测定，俗称Bradford法1，被广泛使用，因为其快速，便捷的协议，以及其相对灵敏度。不幸的是，有很大程度的曲率广泛的蛋白浓度（图1）。因此，只有一个比较高的蛋白浓度2-10 mg / ml的BSA，的狭窄的范围是用于校准曲线，更好地符合线性回归（图1，绿色）。然而，非线性需要未知样品的蛋白质浓度下降校准曲线的有限范围内，以避免大的错误，同时也降低了有限范围内的准确性。非线性呈现一个严重的问题，特别是微克量的蛋白质是不可用时，往往需要多个未知样品稀释。 布拉德福德在原来的文件指出，“非线性的来源是试剂本身，因为是在两个不同颜色的染料形式的的频谱重叠。 “1。事实上，三种形式的考马斯亮蓝染料中的酸碱平衡，在平时的酸性pH值的测定2。红色，蓝色，绿色的形式吸光度最大值在470，590和650 nm，分别为（图2）。蓝色是结合蛋白的形式，形成一个复杂的，强烈的吸收在594 nm的3，4（图2）的光。布拉德福德还指出，“试剂的背景值是不断下降，随着越来越多的的染料是必然蛋白质”1（图3）。因此，我们试图计算减少590纳米背景，增加蛋白质数量增加，通过测量其吸光度在450 nm处的变化，不吸收蛋白染料复杂。我们发现，减少的背景部分，但不充分，占非线性（图4）。 然后，我们推测，免费染料浓度下降，产生另一种失真的线性响应，因为蛋白质的染料结合平衡 5，复合物的形成，不仅取决于蛋白质的浓度，而且还免费染料（图5）。考虑到这两个问题相关的变量自由染料浓度，我们开发了一个数学方程，它描述了蛋白浓度和吸光度测量超过450纳米（图6），590纳米的比例之间的线性关系。的理论和实验研究的详细描述，可以发现在1996年出版分析生物化学6。的数学公式进行了实验测试，结果发现，以产生一个线性校准曲线在整个蛋白的浓度范围（图7）。此外，该方程进行了验证，也由一个独立的Y轴截距 6正确的pH依赖型价值的决心。 详细的协议，为改良的Bradford蛋白含量，用酶标仪吸光度读者： <ol><li>准备的标准，牛血清白蛋白0.1毫克/毫升的原液。任何其他标​​准，可以选择，但请注意，必须在所有实验都使用相同的标准。 </li><li>在去离子水稀释的未知样品。目的是5-50微克/毫升。然而，较高或较低的蛋白浓度是可以接受的，因为没有明显的限制检测的线性范围。然而，必须测量吸光度读取器的线性范围内。 </li><li>淡化布拉德福德试剂（Bio - Rad公司）在去离子水的2.5倍。 </li><li>添加0和10-50 BSA储备液，以一式三份井（0-5微克BSA的校准曲线）液。与去离子水补达到100μL/孔。 </li><li>在不同的井，加100μL未知样品一式三份。可用于未知样品的不同浓度，以提高准确性。 </li><li>加入100μL的稀释布拉德福德试剂所有油井。总成交量为200μL/孔。 </li><li>等待至少5分钟，但不超过60分钟的彩色发展。 </li><li>空白必须是200μL去离子水（而不是零的蛋白质以及染料） 。 </li><li>在590 nm处，并在450 nm处测量吸光度。 </li><li>准备除以590 nm处的吸光度值净额，并在450 nm校准图。注意零蛋白（染料）值应该作为一个数据点（图8）。 </li><li>计算基础上的校准曲线（图9）的线性方程未知样品的浓度。 </li></ol>代表性的成果： 不同的是单一波长在590 nm处的吸光度值超过450纳米，590纳米的比例的吸光度，线性瓦特第i个蛋白质浓度（图10）。 未知样品的蛋白浓度可能只是使用的校准曲线（图10，方程）的线性方程计算。 然而，获得更高的精度是通过测量几个未知样品稀释。为此，准备两个图形。第一个是与微克的X轴（图11，右）的蛋白质标准校正曲线。第二个图是未知样品，与原来的X轴（图11，左）未经稀释的样品液。染料只值应包括在这两个图表（图11）。未知样品的蛋白质含量是派生除以未知样品和标准的斜坡（图11，方程）。 <img src="/files/ftp_upload/1918/1918fig2.jpg" alt="图1" /> 图1。常规布拉德福德校准曲线。线性范围为代表的绿色符号。 <img src="/files/ftp_upload/1918/1918fig1.jpg" alt="图2" /> 图2单独的复杂的蛋白染料和染料的光谱。 <img src="/files/ftp_upload/1918/1918fig3.jpg" alt="图3" /> 图3。蛋白染料的平衡。 <img src="/files/ftp_upload/1918/1918fig4.jpg" alt="图4" /> 图4。减少计算正确的背景部分跌幅非线性。 <img src="/files/ftp_upload/1918/1918fig5.jpg" alt="图5" /> 图5。蛋白染料平衡。 <img src="/files/ftp_upload/1918/1918fig6.jpg" alt="图6" /> 图6。布拉德福蛋白检测的线性化的数学基础。 <img src="/files/ftp_upload/1918/1918fig7.jpg" alt="图7" /> 图7。布拉德福德校准曲线的线性化。 <img src="/files/ftp_upload/1918/1918fig8.jpg" alt="图8" /> 图8。吸光度比值值的计算。 <img src="/files/ftp_upload/1918/1918fig9.jpg" alt="图9" /> 图9线性布拉德福德校准曲线。 <img src="/files/ftp_upload/1918/1918fig10.jpg" alt="图10" /> 图10。布拉德福德校准曲线的线性化。 <img src="/files/ftp_upload/1918/1918fig11.jpg" alt="图11" /> 图11未知样品的浓度计算。

<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>

Bradford蛋白测定是受欢迎的，由于其性能和相对灵敏度缓解。在整个范围内通过协议获得的蛋白质浓度的线性化提出了进一步简化检测，作为未知样品不需要校准曲线的范围内下降。 更重要的是，进一步改进协议在原布拉德福德协议提供以下优点： <ol><li>提高准确度。 </li><li>灵敏度提高约一个量级，从而有可能确定在微孔板检测的牛血清白蛋白 6低至50纳克。 </li>...

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<td>Bradford reagent</td>
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linearization of the bradford protein assay

微克数量在布拉德福德考马斯亮蓝测定蛋白质的测定是通过在590 nm处测定吸光度。最常见的检测，使蛋白质在细胞裂解液，细胞组分或重组蛋白样品的快速和简单的量化，生化测定正常化的目的。然而，一种内在的非线性妥协这种方法的灵敏度和准确性。结果表明，在标准试验条件下，在590 nm处的吸光度测量和450 nm的比例是严格蛋白质浓度的线性。这个简单的程序，提高了精度和提高约10倍，允许定量下降到50吴牛血清白蛋白的检测灵敏度。此外，常用的洗涤剂，用于创建的细胞裂解液引入的干扰大大减少了新的协议。线性方程的集体行动和比尔定律的基础上开发的，完全符合实验数据。

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

Linearization of the Bradford Protein Assay

受到损害的内在非线性的快速，便捷的Bradford法测定蛋白质的准确性和灵敏度。我们展示一个简单的的线性过程，大大提高了准确性，提高约10倍的检测灵敏度，并显着减少洗涤剂干扰。

Bradford蛋白含量的线性

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 次观看.  Tel Aviv University. 受到损害的内在非线性的快速，便捷的Bradford法测定蛋白质的准确性和灵敏度。我们展示一个简单的的线性过程，大大提高了准确性，提高约10倍的检测灵敏度，并显着减少洗涤剂干扰。

视频: 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.

肌动蛋白共沉淀测定;​​ F -肌动蛋白结合蛋白分析

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

科研

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.

蛋白泛素化的检测

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.

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

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.

液相恢复GELFREE 8100分馏系统使用基于分子量分馏

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)

双分子荧光互补（附设）检测蛋白质相互作用的洋葱细胞使用的Helios基因枪

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.

液相亲和捕获试验用磁珠研究蛋白质相互作用：脊髓灰质炎病毒的Nanobody范例

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.

链接捕食风险，草食动物生理应激和植物凋落物的微生物分解

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.

记者的增长分析的蛋白质降解的系统分析

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.

定量免疫检测，以衡量在中心体的变异蛋白水平

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.

凝胶电泳迁移分析（EMSA）为RNA，蛋白质相互作用的研究：IRE / IRP示例

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