| In vitro Microtubule Binding Assay and Dissociation Constant Estimation
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Author:
Date:
2016-03-20
[Abstract] Microtubules (MTs) support an astonishing set of versatile cellular functions ranging from cell division, vesicle transport, and cell and tissue morphogenesis in various organisms. This versatility is in large mediated by MT-associated proteins (MAPs). The neuronal MAP Tau, for example, is stabilizing MTs in axons of the vertebrate nervous system and thus provides the basis for enduring axonal transport and the long life span of neurons (Mandelkow et al., 1994). Tau has been shown to bind to MTs directly in vitro and also to promote their nucleation from α-/β-tubulin subunits (Goode et al., 1994). Recently, we identified a plant-specific protein family called “companion of cellulose synthase” (CC), which was shown to bind MTs and enhance dynamics of the cortical ...
[摘要] 微管（MT）支持惊人的一套多功能细胞功能，从细胞分裂，囊泡运输，以及细胞和组织形态发生在各种生物体。这种多功能性大量由MT相关蛋白（MAP）介导。例如，神经元MAP Tau是稳定脊椎动物神经系统轴突中的MT，因此为持久的轴突运输和神经元的长寿命提供了基础（Mandelkow等人，1994）。 Tau已经显示在体外直接结合MT，并且促进它们从α-/β-微管蛋白亚基的成核（Goode等人，1994）。最近，我们鉴定了称为"纤维素合酶的伴侣"（CC）的植物特异性蛋白质家族，其被证明结合MT并在盐胁迫下增强植物细胞中皮质MT阵列的动力学（Endler等，2015）。因此，CC被假定为帮助植物细胞应对胁迫条件，从而在不利生长条件下维持生物量生产。在这里，我们提供详细的实验信息在体外 MT结合测定，其允许评估感兴趣的蛋白质是否绑定到MT。该测定法在自旋向下方法中利用高分子量的MT，并且使得能够测定解离常数K d sub，其是蛋白质与MT的结合强度的量度。
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| Pot Level Drought Stress Tolerance Assay in Tobacco through Plant Phenotyping and Antioxidant Assay
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Author:
Date:
2015-10-05
[Abstract] Drought is an important abiotic factor which has a huge detrimental impact on crop productivity. Study of plant responses towards drought stress and investigating the mechanism of drought tolerance is crucial for achieving the target of developing drought-tolerant plants. Phenotyping is a cost effective approach which can be adopted to evaluate the severity of drought stress in a plant. Next to phenotyping parameters, biochemical parameters such as the study of antioxidant enzyme activity play significant roles in assessing the extent of drought stress caused injury in a plant. Among the antioxidant enzymes, ascorbate peroxidase is an enzyme which plays a crucial role in drought tolerance in plants. It has been well established that the activity of this enzyme increases under drought ...
[摘要] 干旱是一个重要的非生物因素，对作物生产力有巨大的不利影响。研究植物对干旱胁迫的反应和研究干旱耐受机制对于实现发展耐旱植物的目标是至关重要的。表型是一种成本有效的方法，可用于评价植物中干旱胁迫的严重性。除了表型参数之外，生物化学参数例如抗氧化酶活性的研究在评估植物中干旱胁迫引起的损伤的程度中起重要作用。在抗氧化酶中，抗坏血酸过氧化物酶是在植物的耐旱性中起关键作用的酶。已经确定该酶的活性在干旱胁迫下增加。在这里，我们提出了一个简单和可重复的协议，以调查烟草植物对干旱胁迫通过测量表型参数和抗氧化酶活性的反应。虽然，这些实验已经进行了烟草植物，这个协议可以采用其他作物品种。
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| Bradford Protein Assay
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Author:
Date:
2011-03-20
[Abstract] The Bradford protein assay is used to measure the concentration of total protein in a sample. The principle of this assay is that the binding of protein molecules to Coomassie dye under acidic conditions results in a color change from brown to blue. This method actually measures the presence of the basic amino acid residues, arginine, lysine and histidine, which contributes to formation of the protein-dye complex. Unlike the BCA assay, reducing agents (i.e., DTT and beta—mercaptoethanol) and metal chelators (i.e., EDTA, EGTA) at low concentration do not cause interference. However, the presence of SDS even at low concentrations can interfere with protein-dye binding. This technique was invented by Bradford (1976).
[摘要] Bradford蛋白测定用于测量样品中总蛋白的浓度。 该测定的原理是在酸性条件下蛋白质分子与考马斯染料的结合导致从棕色到蓝色的颜色变化。 该方法实际上测量了有助于形成蛋白质 - 染料复合物的碱性氨基酸残基，精氨酸，赖氨酸和组氨酸的存在。 与BCA测定不同，低浓度的还原剂（即DTT和β-巯基乙醇）和金属螯合剂（即EDTA，EGTA）不引起干扰。 然而，甚至在低浓度的SDS的存在可以干扰蛋白质 - 染料结合。
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