东南亚承认杜克捐赠特别倡议基金, 富国法戈基金, PhRMA 基金会的支持, 以及北卡罗来纳州大学夏洛特的启动资金。

1. 合成 BSA-Au (III.) 络合物
<ol><li>用1毫升高效液相色谱 (HPLC) 级水在5毫升反应瓶中溶解25毫克 BSA。 注意: 该解决方案应该看起来很清楚。</li>
	<li>将金 (III) 氯化三水 (四氯金酸) 溶解于 HPLC 级水中5毫米的浓度。 注意: 该解决方案应显示为黄色。在这种浓度下制备的四氯金酸溶液将导致 BSA 的金比为1:13。<ol>
<li>或者, 在 HPLC 级水中制备溶液, 其浓度介于0.38 毫米 (bsa: au = 1:1) 到20毫米 (bsa: au = 1:50) 之间的四氯金酸。 注: BSA 与金的不同比值将导致激发发射图的红色荧光模式截然不同。</li>
	</ol>
</li>
	<li>将 BSA 的反应瓶放在37摄氏度的水浴中, 用磁力搅拌器大力搅拌 750 rpm。</li>
	<li>搅拌开始后立即加入1毫升的四氯金酸溶液。解决方案的颜色应从透明转换为黄色。</li>
	<li>搅拌混合物2分钟在37°c 和 750 rpm 使用磁力搅拌器。</li>
	<li>进入反应瓶, 添加 100 ul 1 米氢氧化钠的解决方案, 使 pH 值为12。 注: 在添加氢氧化钠后, 溶液应略微变暗至黄褐色, 然后再转回黄色。</li>
	<li>继续搅拌在 750 rpm 2 小时和37摄氏度。解决方案应该慢慢地从黄色变成深黄色/褐色的颜色。这种颜色变化表明红荧光 BSA (III.) 复合物的形成。</li>
	<li>允许样品在室温下坐2天, 溶液将继续变暗到琥珀色的褐色, 荧光强度会增加。<ol>
<li>或者, 让样品继续搅拌37摄氏度12多 h, 因为溶液的颜色演变为琥珀色褐色。</li>
	</ol>
</li>
</ol>2. 合成 BSA-铜 (II)-Au (III)
<ol><li>在1毫升的 HPLC 级水中溶解25毫克 BSA。解决方案应该看起来很清楚。</li>
	<li>将氯化铜 (II) 二水合物溶解于 HPLC 级水中, 浓度为5毫米。该解决方案应显示为浅蓝色。</li>
	<li>添加1毫升的水 BSA 溶液到5毫升反应瓶和地方在水浴在37°c。搅拌 750 rpm 的混合物。</li>
	<li>立即在反应瓶中加入0.5 毫升氯化铜 (II) 溶液, 混合2分钟。溶液将保持浅蓝色。</li>
	<li>添加 75 ul 1 米氢氧化钠, 使 pH 值为 12, 并允许混合2小时。解决方案将变为紫色。</li>
	<li>将四氯金酸溶于 HPLC 级水中, 浓度为5毫米。</li>
	<li>在反应瓶中加入0.5 毫升的水四氯金酸, 用1米氢氧化钠将 pH 值调整回12。</li>
	<li>搅拌反应混合物2小时。 注意: 该解决方案应演变为褐色颜色。</li>
</ol>3. 合成 BSA 镍 (II)-Au (III)
<ol><li>在1毫升的 HPLC 级水中溶解25毫克 BSA。解决方案应该看起来很清楚。</li>
	<li>将氯化镍 (II) 六水合物溶于 HPLC 级水中, 浓度为5毫米。该解决方案应显示为浅绿色。</li>
	<li>添加1毫升的水 BSA 溶液到5毫升反应瓶和地方在水浴在 37 oc. 搅拌混合物在 750 rpm。</li>
	<li>立即在反应瓶中加入0.5 毫升氯化镍 (II) 六水合物溶液, 混合2分钟。 注: 溶液将保持浅绿色。</li>
	<li>添加 75 ul 1 米氢氧化钠, 使 pH 值为 12, 并允许混合2小时。 注意: 溶液将变成深黄色。</li>
	<li>将四氯金酸溶于 HPLC 级水中, 浓度为5毫米。</li>
	<li>在反应瓶中加入0.5 毫升的水四氯金酸, 并将 pH 值调整回12。</li>
	<li>搅拌反应混合物2小时。 注意: 该解决方案应演变为褐色颜色。</li>
</ol>4. [Cys34]-Au (III.) 的合成
<ol><li>溶解2毫克的 n-ethylmaleimide (NEM) 在1毫升磷酸盐缓冲盐水 (PBS, pH 值 7.4)。</li>
	<li>将2毫克的 BSA 溶于1毫升的 PBS-NEM 溶液中。</li>
	<li>将溶液转移到5毫升反应瓶中, 在20摄氏度处搅拌500转1小时。</li>
	<li>透析的解决方案使用 12 kDa 透析油管在500毫升的 PBS, 搅拌 50 rpm 与磁力搅拌器过夜, 以消除反应 NEM。</li>
	<li>在 PBS 中溶解四氯金酸, 浓度为0.4 毫米。 注意: 解决方案将是一个微弱的黄色。</li>
	<li>将反应瓶转移到37摄氏度的水浴中。搅拌750转。</li>
	<li>立即加入1毫升的四氯金酸溶液的反应瓶, 并允许混合2分钟。</li>
	<li>添加 75 ul 1 米氢氧化钠的反应瓶, 使 pH 值为 12, 并允许混合2小时。</li>
</ol>5. 合成 [全硫醇上限-BSA]-Au (III.)
<ol><li>在 HPLC 级水中制备2米尿素和50毫米碳酸氢铵 (NH4HCO3, pH 8.0) 溶液。</li>
	<li>在上述溶液的1毫升中溶解3.3 毫克 BSA, 并转移到5毫升反应瓶。</li>
	<li>用0.25 米 (2-羧乙基) 膦 (TCEP) 的溶液, 将62.5 毫克的 TCEP 溶解在1毫升的 HPLC 水中。</li>
	<li>将 TCEP 的库存溶液添加到反应瓶中, 直到 TCEP 的最终浓度为8毫米。</li>
	<li>在50摄氏度的水浴中孵化溶液1小时。使用磁力搅拌器搅拌 500 rpm。</li>
	<li>让溶液完全冷却到室温。</li>
	<li>用1毫升的 HPLC 级水溶出12.5 毫克 NEM, 制备100毫米 NEM 的库存溶液。</li>
	<li>将 NEM 的库存溶液添加到反应瓶中, 直到 NEM 的最终浓度为16毫米。</li>
	<li>允许溶液结合2小时, 在20摄氏度。搅拌500转。</li>
	<li>透析的解决方案使用 12 kDa 透析油管, 搅拌解决方案在 50 rpm 与磁搅拌器隔夜500毫升50毫米 NH4HCO3 , 以消除过剩的 TCEP, NEM 和尿素。</li>
	<li>将反应瓶移动到37摄氏度的水浴中, 并在750转每分钟搅拌。</li>
	<li>将四氯金酸溶于 HPLC 级水中, 浓度为0.66 毫米。</li>
	<li>立即加入1毫升的四氯金酸溶液的反应瓶, 并允许混合2分钟。</li>
	<li>加入1米氢氧化钠, 直到溶液的 pH 值为 12, 并允许溶液继续混合2小时。</li>
</ol>

<ol>
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作者没有什么可透露的。

在 pH 值为12的白蛋白 (III.) 化合物中制备的红色荧光在λem的发射波长 = 640 毫微米当激发与紫外 (紫外) 光λ前= 365 毫微米 (图 1A)。红色荧光的出现是一个缓慢的过程, 将需要几天在室温下增加到最大强度。在37摄氏度的反应将产生最佳的结果, 虽然更高的温度可以用来更快地产生红色荧光。蛋白质的不可逆转的退化可能发生在温度在45°c23</su...

一种白蛋白-金化合物, 呈紫外 (紫外) 兴奋的红色荧光, 具有显著的斯托克斯移位, 最初是由谢等 al合成的。1. 独特和稳定的红色荧光可以在诸如传感2、3、4、成像5、6、7或纳米医学8等领域找到各种应用. ,9,10,11,12,13。近年来,14、15、16等纳米科学领域的研究人员对此化合物进行了广泛的研究。BSA-au 化合物被解释为 au25簇。所提出方法的目的是对该化合物进行详细的研究, 了解红荧光的起源。通过采用所提出的方法, 可以说明多个 au 结合点的存在, 以及荧光的起源, 替代非盟25簇的单点核。同样的方法也可以用来研究其他蛋白质17,18,19与 Au (III) 的复合物如何改变它们的内在荧光特性。
红荧光 bsa-au 化合物的合成需要对 bsa 的摩尔比 (bsa: au) 进行狭义的控制, 以最大限度地提高荧光强度和激发发射图 (EEM)20中峰值的位置。可以证明, 有多个绑定站点存在于 Au (III) 上, 包括天门冬片段 (或 Asp 片段, 在 BSA N 终点的前四个氨基酸残留物)21,22。34次氨基酸的 BSA (Cys-34) 也显示, 以协调 Au (iii) 和参与的机制, 红色荧光 ([Cys34 上限-BSA]-Au (iii))20。在裂解所有的胱氨酸-胱氨酸二硫化物键和上限所有硫醇, 红色荧光不产生 ([全硫醇上限-BSA]-Au (III))。这表明了胱氨酸-胱氨酸二硫化物键作为非盟 (III.) 结合部位产生红色荧光的必要性。
蛋白质化学技术还没有被广泛用于研究纳米科学群落中的 BSA (III.) 配合物。然而, 使用这些技术来了解这些复合体的某些方面, 以及对 BSA 中的非盟 (III) 结合点有详细的了解是很有价值的。本文旨在展示其中的一些技术。

<table>
	<tbody>
		<tr>
			<td>Bovine Serum Albumin (BSA), 96%</td>
			<td>Sigma-Aldrich</td>
			<td>A5611</td>
			<td></td>
		</tr>
		<tr>
			<td>gold (III) chloride trihydrate, 99.9%</td>
			<td>Sigma-Aldrich</td>
			<td>520918</td>
			<td></td>
		</tr>
		<tr>
			<td>Copper (II) chloride dihydrate, 99.999%</td>
			<td>Sigma-Aldrich</td>
			<td>459097</td>
			<td></td>
		</tr>
		<tr>
			<td>Nickel (II) chloride hexahydrate, 99.9%</td>
			<td>Sigma-Aldrich</td>
			<td>654507</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Ethylmaleimide (NEM), &#62;99.0%</td>
			<td>Sigma-Aldrich</td>
			<td>4259</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), &#62;98.0%</td>
			<td>Sigma-Aldrich</td>
			<td>C4706</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide, &#62;98.0%</td>
			<td>Sigma-Aldrich</td>
			<td>S8045</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea, 99.5%</td>
			<td>Chem-Implex Int&#39;l</td>
			<td>30142</td>
			<td></td>
		</tr>
		<tr>
			<td>Phospate buffered saline (PBS)</td>
			<td>Corning</td>
			<td>MT21040CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium bicarbonate, 99.5%</td>
			<td>Sigma-Aldrich</td>
			<td>9830</td>
			<td></td>
		</tr>
	</tbody>
</table>

luminophore formation in various conformations of bovine serum albumin by binding of gold(iii)

所提出的协议的目的是研究金 (iii) 对 bsa 的结合过程, 产生构象变化诱导红荧光 (λem = 640 nm) 的 bsa-Au (iii) 配合物。该方法调整 ph 值, 表明红荧光的出现与 BSA 构象的 ph 诱导的平衡过渡相关。红色荧光 BSA-Au (III.) 配合物只能形成与 pH 值在9.7 或以上的调整, 这对应于 "A 形" 构象的 BSA。介绍了将 BSA 调整为金摩尔比的协议, 并对金 (III.) 结合过程的时间过程进行了监测。每 BSA (III) 的最小数目, 以产生红色荧光, 小于七。我们描述了该协议的步骤, 以说明在 BSA 中存在多个 Au (III) 绑定站点。首先, 通过添加铜 (二) 或镍 (镍 (ii)) 阳离子其次为 au (iii), 这种方法揭示了一个结合点的非盟 (iii), 这不是红色荧光。其次, 通过硫醇封堵剂对 BSA 进行改性, 揭示了另一种 nonfluorophore 形成的金 (III.) 结合部位。第三, 通过对二硫化物键的劈裂和封盖改变 BSA 构象, 说明可能的 Au (III.) 结合点。该协议描述, 以控制 BSA 构象和 Au (III) 结合, 可以广泛应用于研究其他蛋白质和金属阳离子的相互作用。

从 bsa-Au (III.) 络合物的荧光, 观察到, 白蛋白 (λem = 400 nm) 与红色荧光 (λem = 640 nm) 的内在蓝色荧光的转换发生在 pH 9.7 通过平衡转换 (图 1)。EEM (III.) 在不同的 bsa 到金摩尔比率如图 2所示, 这一数据表明, 改变摩尔比产生相同的发射波长在不同的激发波长。Cu (ii)、镍 (ii) 和 Au (III) 在 BSA (<strong class="xfi...

Watch this Scientific Journal Video about Luminophore Formation in Various Conformations of Bovine Serum Albumin by Binding of GoldIII at JoVE.com

Luminophore Formation in Various Conformations of Bovine Serum Albumin by Binding of GoldIII

本文介绍了研究金阳离子 (III.) 对牛血清白蛋白的各种构象和构象依赖性独特的 bsa-Au 荧光的表征的协议。

结合金 (III.) 对牛血清白蛋白不同构象的 Luminophore 形成

The purpose of the presented protocols is to study the process of Au(III) binding to BSA, yielding conformation change-induced red fluorescence (&#955;em ...

luminophore-formation-various-conformations-bovine-serum-albumin

Research

JoVE Journal

Chemistry

Luminophore Formation in Various Conformations of Bovine Serum Albumin by Binding of Gold(III)

6.8K 次观看.  University of North Carolina. 本文介绍了研究金阳离子 (III.) 对牛血清白蛋白的各种构象和构象依赖性独特的 bsa-Au 荧光的表征的协议。

视频: Luminophore Formation in Various Conformations of Bovine Serum Albumin by Binding of GoldIII

LabVIEW-operated Novel Nanoliter Osmometer for Ice Binding Protein Investigations

Ice-binding proteins (IBPs), including antifreeze proteins, ice structuring proteins, thermal hysteresis proteins, and ice recrystallization inhibition proteins, are found in cold-adapted organisms and protect them from freeze injuries by interacting with ice crystals. IBPs are found in a variety of organism, including fish1, plants2, 3, arthropods4, 5, fungi6, and bacteria7. IBPs adsorb to the surfaces of ice crystals and prevent water molecules from joining the ice lattice at the IBP adsorption location. Ice that grows on the crystal surface between the adsorbed IBPs develops a high curvature that lowers the temperature at which the ice crystals grow, a phenomenon referred to as the Gibbs-Thomson effect. This depression creates a gap (thermal hysteresis, TH) between the melting point and the nonequilibrium freezing point, within which ice growth is arrested8-10, see Figure 1. One of the main tools used in IBP research is the nanoliter osmometer, which facilitates measurements of the TH activities of IBP solutions. Nanoliter osmometers, such as the Clifton instrument (Clifton Technical Physics, Hartford, NY,) and Otago instrument (Otago Osmometers, Dunedin, New Zealand), were designed to measure the osmolarity of a solution by measuring the melting point depression of droplets with nanoliter volumes. These devices were used to measure the osmolarities of biological samples, such as tears11, and were found to be useful in IBP research. Manual control over these nanoliter osmometers limited the experimental possibilities. Temperature rate changes could not be controlled reliably, the temperature range of the Clifton instrument was limited to 4,000 mOsmol (about -7.5 &deg;C), and temperature recordings as a function of time were not an available option for these instruments.
We designed a custom-made computer-controlled nanoliter osmometer system using a LabVIEW platform (National Instruments). The cold stage, described previously9, 10, contains a metal block through which water circulates, thereby functioning as a heat sink, see Figure 2. Attached to this block are thermoelectric coolers that may be driven using a commercial temperature controller that can be controlled via LabVIEW modules, see Figure 3. Further details are provided below. The major advantage of this system is its sensitive temperature control, see Figure 4. Automated temperature control permits the coordination of a fixed temperature ramp with a video microscopy output containing additional experimental details.
To study the time dependence of the TH activity, we tested a 58 kDa hyperactive IBP from the Antarctic bacterium Marinomonas primoryensis (MpIBP)12. This protein was tagged with enhanced green fluorescence proteins (eGFP) in a construct developed by Peter Davies' group (Queens University)10. We showed that the temperature change profile affected the TH activity. Excellent control over the temperature profile in these experiments significantly improved the TH measurements. The nanoliter osmometer additionally allowed us to test the recrystallization inhibition of IBPs5, 13. In general, recrystallization is a phenomenon in which large crystals grow larger at the expense of small crystals. IBPs efficiently inhibit recrystallization, even at low concentrations14, 15. We used our LabVIEW-controlled osmometer to quantitatively follow the recrystallization of ice and to enforce a constant ice fraction using simultaneous real-time video analysis of the images and temperature feedback from the sample chamber13. The real-time calculations offer additional control options during an experimental procedure. A stage for an inverted microscope was developed to accommodate temperature-controlled microfluidic devices, which will be described elsewhere16.
The Cold Stage System
The cold stage assembly (Figure 2) consists of a set of thermoelectric coolers that cool a copper plate. Heat is removed from the stage by flowing cold water through a closed compartment under the thermoelectric coolers. A 4 mm diameter hole in the middle of the copper plate serves as a viewing window. A 1 mm diameter in-plane hole was drilled to fit the thermistor. A custom-made copper disc (7 mm in diameter) with several holes (500 &mu;m in diameter) was placed on the copper plate and aligned with the viewing window. Air was pumped at a flow rate of 35 ml/sec and dried using Drierite (W.A. Hammond). The dry air was used to ensure a dry environment at the cooling stage. The stage was connected via a 9 pin connection outlet to a temperature controller (Model 3040 or 3150, Newport Corporation, Irvine, California, US). The temperature controller was connected via a cable to a computer GPIB-PCI card (National instruments, Austin, Texas, USA).

Ice binding proteins (IBPs), also known as antifreeze proteins, inhibit ice growth and are a promising additive for use in the cryopreservation of tissues. The main tool used to investigate IBPs is the nanoliter osmometer. We developed a home-designed cooling stage mounted on an optical microscope and controlled using a custom-built LabVIEW routine. The nanoliter osmometer described here manipulated the sample temperature in an ultra-sensitive manner.

LabVIEW操作的新型纳升渗透压仪冰结合蛋白的调查

Ice-binding proteins (IBPs), including antifreeze proteins, ice structuring proteins, thermal hysteresis proteins, and ice recrystallization inhibition ...

科研

化学

Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS)

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. The approach offers an unbiased and in-depth analysis that can enable the development of diagnostic tests, novel therapies, and further our understanding of disease processes. The inherent chemical diversity of the metabolome creates significant analytical challenges and there is no single experimental approach that can detect all metabolites. Additionally, the biological variation in individual metabolism and the dependence of metabolism on environmental factors necessitates large sample numbers to achieve the appropriate statistical power required for meaningful biological interpretation. To address these challenges, this tutorial outlines an analytical workflow for large scale non-targeted metabolite profiling of serum by UPLC-MS. The procedure includes guidelines for sample organization and preparation, data acquisition, quality control, and metabolite identification and will enable reliable acquisition of data for large experiments and provide a starting point for laboratories new to non-targeted metabolite profiling by UPLC-MS.

Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry UPLC-MS

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. This article outlines a typical workflow utilized for non-targeted metabolite profiling of serum including sample organization and preparation, data acquisition, data analysis, quality control, and metabolite identification.

规模以上非靶向代谢组学剖析血清超高效液相色谱 - 质谱（UPLC-MS）

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to ...

Covalent Binding of BMP-2 on Surfaces Using a Self-assembled Monolayer Approach

Bone morphogenetic protein 2 (BMP-2) is a growth factor embedded in the extracellular matrix of bone tissue. BMP-2 acts as trigger of mesenchymal cell differentiation into osteoblasts, thus stimulating healing and de novo bone formation. The clinical use of recombinant human BMP-2 (rhBMP-2) in conjunction with scaffolds has raised recent controversies, based on the mode of presentation and the amount to be delivered. The protocol presented here provides a simple and efficient way to deliver BMP-2 for in vitro studies on cells. We describe how to form a self-assembled monolayer consisting of a heterobifunctional linker, and show the subsequent binding step to obtain covalent immobilization of rhBMP-2. With this approach it is possible to achieve a sustained presentation of BMP-2 while maintaining the biological activity of the protein. In fact, the surface immobilization of BMP-2 allows targeted investigations by preventing unspecific adsorption, while reducing the amount of growth factor and, most notably, hindering uncontrolled release from the surface. Both short- and long-term signaling events triggered by BMP-2 are taking place when cells are exposed to surfaces presenting covalently immobilized rhBMP-2, making this approach suitable for in vitro studies on cell responses to BMP-2 stimulation.

We describe a method for accomplishing efficient immobilization of BMP-2 on surfaces. Our approach is based on the formation of a self-assembled monolayer to achieve the covalent binding of BMP-2 via its free amine residues. This method is a useful tool to study signaling at the cell membrane.

BMP-2的共价结合利用表面上的自组装单层膜法

Bone morphogenetic protein 2 (BMP-2) is a growth factor embedded in the extracellular matrix of bone tissue. BMP-2 acts as trigger of mesenchymal cell ...

Microwave-assisted Functionalization of Poly(ethylene glycol) and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large variety of PEG molecular weights and configurations (arm number, arm length, and branching pattern) affords researchers tight control over resulting hydrogel structures and properties, including Young&#8217;s modulus and mesh size. This video will illustrate a rapid, efficient, solvent-free, microwave-assisted method to methacrylate PEG precursors into poly(ethylene glycol) dimethacrylate (PEGDM). This synthetic method provides much-needed starting materials for applications in drug delivery and regenerative medicine. The demonstrated method is superior to traditional methacrylation methods as it is significantly faster and simpler, as well as more economical and environmentally friendly, using smaller amounts of reagents and solvents. We will also demonstrate an adaptation of this technique for on-resin methacrylamide functionalization of peptides. This on-resin method allows the N-terminus of peptides to be functionalized with methacrylamide groups prior to deprotection and cleavage from resin. This allows for selective addition of methacrylamide groups to the N-termini of the peptides while amino acids with reactive side groups (e.g.&#160;primary amine of lysine, primary alcohol of serine, secondary alcohols of threonine, and phenol of tyrosine) remain protected, preventing functionalization at multiple sites. This article will detail common analytical methods (proton Nuclear Magnetic Resonance spectroscopy (;H-NMR) and Matrix Assisted Laser Desorption Ionization Time of Flight mass spectrometry (MALDI-ToF)) to assess the efficiency of the functionalizations. Common pitfalls and suggested troubleshooting methods will be addressed, as will modifications of the technique which can be used to further tune macromer functionality and resulting hydrogel physical and chemical properties. Use of synthesized products for the formation of hydrogels for drug delivery and cell-material interaction studies will be demonstrated, with particular attention paid to modifying hydrogel composition to affect mesh size, controlling hydrogel stiffness and drug release.

Microwave-assisted Functionalization of Polyethylene glycol and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

This video will illustrate a rapid, efficient method to methacrylate poly(ethylene glycol), enabling chain polymerizations and hydrogel synthesis. It will demonstrate how to similarly introduce methacrylamide functionalities into peptides, detail common analytical methods to assess functionalization efficiency, provide suggestions for troubleshooting and advanced modifications, and demonstrate typical hydrogel characterization techniques.

保利的微波辅助功能化（乙二醇）和On-树脂链聚合反应，并形成水凝胶的肽使用

One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large ...

Formation of Ordered Biomolecular Structures by the Self-assembly of Short Peptides

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control self-assembly and mimicking this process in vitro will bring about major advances in the areas of materials science and nanotechnology. Among the available biological building blocks, peptides have several advantages as they present substantial diversity, their synthesis in large scale is straightforward, and they can easily be modified with biological and chemical entities1,2. Several classes of designed peptides such as cyclic peptides, amphiphile peptides and peptide-conjugates self-assemble into ordered structures in solution. Homoaromatic dipeptides, are a class of short self-assembled peptides that contain all the molecular information needed to form ordered structures such as nanotubes, spheres and fibrils3-8. A large variety of these peptides is commercially available.
This paper presents a procedure that leads to the formation of ordered structures by the self-assembly of homoaromatic peptides. The protocol requires only commercial reagents and basic laboratory equipment. In addition, the paper describes some of the methods available for the characterization of peptide-based assemblies. These methods include electron and atomic force microscopy and Fourier-Transform Infrared Spectroscopy (FT-IR). Moreover, the manuscript demonstrates the blending of peptides (coassembly) and the formation of a &#34;beads on a string&#34;-like structure by this process.9 The protocols presented here can be adapted to other classes of peptides or biological building blocks and can potentially lead to the discovery of new peptide-based structures and to better control of their assembly.

This paper describes the formation of highly ordered peptide-based structures by the spontaneous process of self-assembly. The method utilizes commercially available peptides and common lab equipment. This technique can be applied to a large variety of peptides and may lead to the discovery of new peptide-based assemblies.

生物分子有序结构由短肽的自组装形成

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control ...

Determining the Ice-binding Planes of Antifreeze Proteins by Fluorescence-based Ice Plane Affinity

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice through their ice-binding surfaces. Fluorescence-based ice plane affinity (FIPA) analysis is a modified technique used to determine the ice planes to which the AFPs bind. FIPA is based on the original ice-etching method for determining AFP-bound ice-planes. It produces clearer images in a shortened experimental time. In FIPA analysis, AFPs are fluorescently labeled with a chimeric tag or a covalent dye then slowly incorporated into a macroscopic single ice crystal, which has been preformed into a hemisphere and oriented to determine the a- and c-axes. The AFP-bound ice hemisphere is imaged under UV light to visualize AFP-bound planes using filters to block out nonspecific light. Fluorescent labeling of the AFPs allows real-time monitoring of AFP adsorption into ice. The labels have been found not to influence the planes to which AFPs bind. FIPA analysis also introduces the option to bind more than one differently tagged AFP on the same single ice crystal to help differentiate their binding planes. These applications of FIPA are helping to advance our understanding of how AFPs bind to ice to halt its growth and why many AFP-producing organisms express multiple AFP isoforms.

Antifreeze proteins (AFPs) bind to specific planes of ice to prevent or slow ice growth. Fluorescence-based ice plane affinity (FIPA) analysis is a modification of the original ice-etching method for determination of AFP-bound ice planes. AFPs are fluorescently labeled, incorporated into macroscopic single ice crystals, and visualized under UV light.

通过荧光基冰平面亲和力确定防冻蛋白的冰结合平面

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice ...

Water in Oil Emulsions: A New System for Assembling Water-soluble Chlorophyll-binding Proteins with Hydrophobic Pigments

Chlorophylls (Chls) and bacteriochlorophylls (BChls) are the primary cofactors that carry out photosynthetic light harvesting and electron transport. Their functionality critically depends on their specific organization within large and elaborate multisubunit transmembrane protein complexes. In order to understand at the molecular level how these complexes facilitate solar energy conversion, it is essential to understand protein-pigment, and pigment-pigment interactions, and their effect on excited dynamics. One way of gaining such understanding is by constructing and studying complexes of Chls with simple water-soluble recombinant proteins. However, incorporating the lipophilic Chls and BChls into water-soluble proteins is difficult. Moreover, there is no general method, which could be used for assembly of water-soluble proteins with hydrophobic pigments. Here, we demonstrate a simple and high throughput system based on water-in-oil emulsions, which enables assembly of water-soluble proteins with hydrophobic Chls. The new method was validated by assembling recombinant versions of the water-soluble chlorophyll binding protein of Brassicaceae plants (WSCP) with Chl a. We demonstrate the successful assembly of Chl a using crude lysates of WSCP expressing E. coli cell, which may be used for developing a genetic screen system for novel water-soluble Chl-binding proteins, and for studies of Chl-protein interactions and assembly processes.

This manuscript describes a simple and high-throughput method for assembling water-soluble proteins with hydrophobic pigments that is based on water-in-oil emulsions. We demonstrate the effectiveness of the method in the assembly of native chlorophylls with four variants of recombinant water-soluble-chlorophyll binding proteins (WSCPs) of Brassica plants expressed in E. coli.

水在油乳剂:与疏水性颜料装配水溶性叶绿素结合蛋白的新系统

Chlorophylls (Chls) and bacteriochlorophylls (BChls) are the primary cofactors that carry out photosynthetic light harvesting and electron transport. ...

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular identity and function. Each of the seven PtdInsPs varies in their distribution and abundance, which are tightly regulated by specific kinases and phosphatases. The abundance of PtdInsPs can change abruptly in response to various signaling events or disturbance of the regulatory machinery. To understand how these events lead to changes in the amount of PtdInsPs and their resulting impact, it is important to quantify PtdInsP levels before and after a signaling event or between control and abnormal conditions. However, due to their low abundance and similarity, quantifying the relative amounts of each PtdInsP can be challenging. This article describes a method for quantifying PtdInsP levels by metabolically labeling cells with 3H-myo-inositol, which is incorporated into PtdInsPs. Phospholipids are then precipitated and deacylated. The resulting soluble 3H-glycero-inositides are further extracted, separated by high-performance liquid chromatography (HPLC), and detected by flow scintillation. The labeling and processing of yeast samples is described in detail, as well as the instrumental setup for the HPLC and flow scintillator. Despite losing structural information regarding acyl chain content, this method is sensitive and can be optimized to concurrently quantify all seven PtdInsPs in cells.

Phosphoinositides are signaling lipids whose relative abundance rapidly changes in response to various stimuli. This article describes a method to measure the abundance of phosphoinositides by metabolically labeling cells with 3H-myo-inositol, followed by extraction and deacylation. Extracted glycero-inositides are then separated by high-performance liquid chromatography and quantified by flow scintillation.

放射性标记和磷酸肌醇的细胞水平的定量高效液相色谱耦合流动闪烁

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular ...

In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS

This work demonstrates in situ characterization of protein biomolecules in the aqueous solution using the System for Analysis at the Liquid Vacuum Interface (SALVI) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). The fibronectin protein film was immobilized on the silicon nitride (SiN) membrane that forms the SALVI detection area. During ToF-SIMS analysis, three modes of analysis were conducted including high spatial resolution mass spectrometry, two-dimensional (2D) imaging, and depth profiling. Mass spectra were acquired in both positive and negative modes. Deionized water was also analyzed as a reference sample. Our results show that the fibronectin film in water has more distinct and stronger water cluster peaks compared to water alone. Characteristic peaks of amino acid fragments are also observable in the hydrated protein ToF-SIMS spectra. These results illustrate that protein molecule adsorption on a surface can be studied dynamically using SALVI and ToF-SIMS in the liquid environment for the first time.

In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS

This work presents a protocol for liquid handling and sample introduction to a microchannel for in situ time-of-flight secondary ion mass spectrometry analysis of protein biomolecules in an aqueous solution.

在水中水合蛋白原位表征SALVI和TOF-SIMS

This work demonstrates in situ characterization of protein biomolecules in the aqueous solution using the System for Analysis at the Liquid Vacuum ...

DNA-magnetic Particle Binding Analysis by Dynamic and Electrophoretic Light Scattering

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the evaluation of DNA-magnetic particles binding via dynamic light scattering (DLS) and electrophoretic light scattering (ELS). Analysis by DLS provides valuable information on the physicochemical properties of particles including particle size, polydispersity, and zeta potential. The latter describes the surface charge of the particle which plays major role in electrostatic binding of materials such as DNA. Here, a comparative analysis exploits three chemical modifications of nanoparticles and microparticles and their effects on DNA binding and elution. Chemical modifications by branched polyethylenimine, tetraethyl orthosilicate and (3-aminopropyl)triethoxysilane are investigated. Since DNA exhibits a negative charge, it is expected that zeta potential of particle surface will decrease upon binding of DNA. Forming of clusters should also affect particle size. In order to investigate the efficiency of these particles in isolation and elution of DNA, the particles are mixed with DNA in low pH (~6), high ionic strength and dehydration environment. Particles are washed on magnet and then DNA is eluted by Tris-HCl buffer (pH = 8). DNA copy number is estimated using quantitative polymerase chain reaction (PCR). Zeta potential, particle size, polydispersity and quantitative PCR data are evaluated and compared. DLS is an insightful and supporting method of analysis that adds a new perspective to the process of screening of particles for DNA isolation.

This protocol describes the synthesis of magnetic particles and evaluation of their DNA-binding properties via dynamic and electrophoretic light scattering. This method focuses on monitoring changes in particle size, their polydispersity and zeta potential of particle surface which play major role in binding of materials such as DNA.

动态和电泳光散射的 DNA-磁性颗粒结合分析

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the ...