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/pubmed?term=((A+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding%5BTitle%5D)+AND+%22Anal+Biochem%22%5BJournal%5D)">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/pubmed?term=((A+spectral+study+of+the+charge+forms+of+Coomassie+blue+G%5BTitle%5D)+AND+%22Anal+Biochem%22%5BJournal%5D)">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/pubmed?term=((A+comparison+of+the+binding+of+Coomassie+brilliant+blue+to+proteins+at+low+and+neutral+pH%5BTitle%5D)+AND+%22Anal+Biochem%22%5BJournal%5D)">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/pubmed?term=((Mechanism+of+dye+response+and+interference+in+the+Bradford+protein+assay%5BTitle%5D)+AND+%22Anal+Biochem%22%5BJournal%5D)">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/pubmed?term=((The+binding+interaction+of+Coomassie+blue+with+proteins%5BTitle%5D)+AND+%22Anal+Biochem%22%5BJournal%5D)">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/pubmed?term=((Linearization+of+the+Bradford+protein+assay+increases+its+sensitivity%3A+theoretical+and+experimental+studies%5BTitle%5D)+AND+%22Anal+Biochem%22%5BJournal%5D)">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><li>提高了灵敏度，使在场的洗涤剂蛋白质定量。随着原布拉德福德协议，通过干扰细胞裂解通常使用的洗涤剂，往往会导致蛋白质在反应减弱。的灵敏度提高一个量级，使样品稀释到一个点，消除干扰是由洗涤剂。请注意，标准样品应包含在相同的洗涤剂的成分和浓度稀释的未知样品。 </li></ol>蛋白染料复杂和自由的红色染料形式的吸收峰分别在594和470 nm，。建议在590-600纳米测量吸光度，以达到最大的蛋白浓度变化的敏感性，并在450-485纳米，以确保线性。 由于光学路径长度考虑，更高的精度可达到1毫升的检测试管放在一个普通的分光光度计，而不是酶标仪。在这种情况下，规模高达5倍的卷。 这绝对是强制性的，去离子水将用于为空白。线性校正曲线无法取得零的蛋白质标准含染料样品为空白。

<table><tbody><tr><td>Bradford reagent</td><td>Bio-Rad</td><td>500-0006</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Amersco</td><td>0332</td><td></td></tr><tr><td>Multiplate absorbance reader</td><td>BioTek</td><td>Synergy2</td><td></td></tr></tbody></table>

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

Universidad San Sebastian

Research

JoVE Journal

Biology

99.8K 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

Bio-layer Interferometry for Measuring Kinetics of Protein-protein Interactions and Allosteric Ligand Effects

We describe the use of Bio-layer Interferometry to study inhibitory interactions of subunit &#949;&#160;with the catalytic complex of Escherichia coli&#160;ATP synthase. Bacterial F-type ATP synthase is the target of a new, FDA-approved antibiotic to combat drug-resistant tuberculosis. Understanding bacteria-specific auto-inhibition of ATP synthase by the C-terminal domain of subunit &#949;&#160;could provide a new means to target the enzyme for discovery of antibacterial drugs. The C-terminal domain of &#949;&#160;undergoes a dramatic conformational change when the enzyme transitions between the active and inactive states, and catalytic-site ligands can influence which of &#949;&#39;s conformations is predominant. The assay measures kinetics of &#949;&#39;s binding/dissociation with the catalytic complex, and indirectly measures the shift of enzyme-bound &#949;&#160;to and from the apparently nondissociable inhibitory conformation. The Bio-layer Interferometry signal is not overly sensitive to solution composition, so it can also be used to monitor allosteric effects of catalytic-site ligands on &#949;&#39;s conformational changes.

The protocols here describe kinetic assays of protein-protein interactions with Bio-layer Interferometry. F-type ATP synthase, which is involved in cellular energy metabolism, can be inhibited by its &#949; subunit in bacteria. We have adapted Bio-layer Interferometry to study interactions of the catalytic complex with &#949;&#8217;s inhibitory C-terminal domain.

生物层干涉测量的蛋白质 - 蛋白质相互作用和变构配体反应动力学的影响

We describe the use of Bio-layer Interferometry to study inhibitory interactions of subunit &#949;&#160;with the catalytic complex of Escherichia ...

科研

化学

Hellgrammite as a Non-Conventional Biomonitor for Surface Water and Environmental Health Assessment

The larvae of dobsonflies from the genus Corydalus, commonly known as Hellgrammites, are characterized by their notable size, extensive range of occurrence, and extended period of immaturity, which can last up to one year. Hellgrammites are clearly known to exhibit sensitivity to pollution and habitat structure impacts. Given these unique features, using Corydalus texanus larvae is highly suitable as reliable biomonitoring agents to assess the ecological integrity of aquatic ecosystems. This protocol aims to provide the necessary tools for C. texanus assessment and demonstrate their efficacy through a case study. Research findings have practical implications, indicating that C. texanus larvae exhibit early-warning responses to mining pollution, bioaccumulating high amounts of heavy metals such as Zn, Fe, and Al. The presence or absence of C. texanus populations may serve as a helpful indicator for identifying potential issues related to ecosystem health. The unconventional approach has shown early warnings of pollution in mining-impacted sites, highlighting the need for timely action to protect the environment. Given their unique traits, the use of C. texanus larvae is highly suggested as a reliable non-conventional bioindicator.

This protocol describes the use of biomarkers for the early detection of deleterious impacts in aquatic ecosystems. The biomarkers are closely related to sentinel traits, and their changes aid in detecting early-warning damages.

Hellgrammite 作为地表水和环境健康评估的非常规生物监测器

The larvae of dobsonflies from the genus Corydalus, commonly known as Hellgrammites, are characterized by their notable size, extensive range of ...

环境科学

智能手机作为使用 Bradford 分析定量蛋白质的分析设备

RGBradford: Protein Quantitation with a Smartphone Camera

Protein quantitation is an essential procedure in life sciences research. Amongst several other methods, the Bradford assay is one of the most used. Because of its widespread, the limitations and advantages of the Bradford assay have been exhaustively reported, including several modifications of the original method to improve its performance. One of the alterations of the original method is the use of a smartphone camera as an analytical instrument. Taking advantage of the three forms of the Coomassie Brilliant Blue dye that exist in the conditions of the Bradford assay, this paper describes how to accurately quantify protein in samples using color data extracted from a single picture of a microplate. After performing the assay in a microplate, a picture is taken using a smartphone camera, and RGB color data is extracted from the picture using a free and open-source image analysis software application. Then, the ratio of blue to green intensity (in the RGB scale) of samples with unknown concentrations of protein is used to calculate the protein content based on a standard curve. No significant difference is observed between values calculated using RGB color data and those calculated using conventional absorbance data.

作者聚焦:使用智能手机通过 Bradford 分析进行蛋白质定量的另一种方法 

This paper provides a protocol for protein quantification using the Bradford assay and a smartphone as an analytical device. Protein levels in samples can be quantified using color data extracted from a picture of a microplate taken with a smartphone.

RGBradford:使用智能手机相机进行蛋白质定量

Protein quantitation is an essential procedure in life sciences research. Amongst several other methods, the Bradford assay is one of the most used. ...

生物化学

使用生物物理测定表征 PROTAC 中三元复合物的形成

The Development and Application of Biophysical Assays for Evaluating Ternary Complex Formation Induced by Proteolysis Targeting Chimeras (PROTACS)

E3 ligases and proteins targeted for degradation can be induced to form complexes by heterobifunctional molecules in a multi-step process. The kinetics and thermodynamics of the interactions involved contribute to efficiency of ubiquitination and resulting degradation of the protein. Biophysical techniques such as surface plasmon resonance (SPR), biolayer interferometry (BLI), and isothermal titration calorimetry (ITC) provide valuable information that can be used in the optimization of those interactions. Using two model systems, a biophysical assay tool kit for understanding the cooperativity of ternary complex formation and the impact of the &#39;hook effect&#39; on binding kinetics was established. In one case, a proteolysis targeting chimera (PROTAC) molecule that induced ternary complex formation between Brd4BD2 and VHL was evaluated. The heterobifunctional molecule, MZ1, has nM affinities for both the Brd4BD2 protein (SPR KD = 1 nM, ITC KD = 4 nM) and the VHL complex (SPR KD = 29 nM, ITC KD = 66 nM). For this system, robust SPR, BLI, and ITC assays were developed that reproduced published results demonstrating the cooperativity of ternary complex formation. In the other case, a molecule that induced ternary complexes between a 46.0 kDa protein, PPM1D, and cereblon [CRBN (319-442)] was studied. The heterobifunctional molecule, BRD-5110, has an SPR KD = 1 nM for PPM1D but much weaker binding against the truncated CRBN (319-442) complex (SPR KD= ~ 3 &#181;M). In that case, the binding for CRBN in SPR was not saturable, resulting in a &#34;hook-effect&#34;. Throughput and reagent requirements for SPR, BLI, and ITC were evaluated, and general recommendations for their application to PROTAC projects were provided.

作者聚焦：评估用于表征 PROTACS 三元复合物的生物物理检测 

Here we describe protocols for the biophysical characterization of ternary complex formation induced by proteolysis targeting chimeras (PROTACS) that involve the ubiquitin ligases Von Hippel-Lindau E3 ligase (VHL) and Cereblon (CRBN). Biophysical methods illustrated herein include surface plasmon resonance (SPR), biolayer interferometry (BLI), and isothermal titration calorimetry (ITC).

用于评估蛋白水解靶向嵌合体（PROTACS）诱导的三元复合物形成的生物物理测定方法的发展和应用

E3 ligases and proteins targeted for degradation can be induced to form complexes by heterobifunctional molecules in a multi-step process. The kinetics ...

<meta charset="utf8"></head><body>使用无标记方法定量分析 RPA-ssDNA 结合动力学

Analyzing DNA-Protein Interactions with Streptavidin-Based Biolayer Interferometry

Protein-DNA interactions underpin essential cellular processes. Understanding these interactions is critical for elucidating the molecular mechanisms of various pathways. Key factors such as the structure, sequence, and length of a DNA molecule can significantly influence protein binding. Bio-layer interferometry (BLI) is a label-free technique that measures binding kinetics between molecules, offering a straightforward and precise approach to quantitatively study protein-DNA interactions. A major advantage of BLI over traditional gel-based methods is its ability to provide real-time data on binding kinetics, enabling accurate measurement of the equilibrium dissociation constant (KD) for dynamic protein-DNA interactions. This article presents a basic protocol for determining the KD value of the interaction between a DNA replication protein, replication protein A (RPA), and a single-stranded DNA (ssDNA) substrate. RPA binds ssDNA with high affinity but must also be easily displaced to facilitate subsequent protein interactions within biological pathways. In the described BLI assay, biotinylated ssDNA is immobilized on a streptavidin-coated biosensor. The binding kinetics (association and dissociation) of RPA to the biosensor-bound DNA are then measured. The resulting data are analyzed to derive precise values for the association rate constant (ka), dissociation rate constant (kd), and equilibrium binding constant (KD) using system software.

<meta charset="utf8"></head><body>使用基于链霉亲和素的生物膜干涉测量法分析 DNA-蛋白质相互作用

This article describes a protocol for studying DNA-protein interactions using a streptavidin-based biolayer interferometry (BLI) system. It outlines the essential steps and considerations for utilizing either basic or advanced binding kinetics to determine the equilibrium binding affinity (KD) of the interaction.

使用基于链霉亲和素的生物膜干涉测量法分析 DNA-蛋白质相互作用

Protein-DNA interactions underpin essential cellular processes. Understanding these interactions is critical for elucidating the molecular mechanisms of ...

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