東南は、シャーロットからデューク特別なイニシアチブ基金、ウェルズ ファーゴ基金 PhRMA 財団、スタートアップ資金、ノースカロライナの大学からの支援を認めています。

1. BSA-Au(III) 複合体の合成
<ol><li>高速液体クロマトグラフィー (HPLC) グレード水 5 mL 反応瓶で 1 mL に BSA の 25 mg を溶解します。 注: ソリューションが明確に表示されます。</li>
	<li>金 (III) 塩化物三水和物 (塩化金酸) を 5 mM HPLC グレードの水の濃度に溶解します。 注: ソリューションは黄色で表示されます。この濃度で塩化金酸溶液は Au 比 1:13、BSA になります。<ol>
<li>また、どこの濃度と塩化金酸の溶液を調製 0.38 ミリメートルの間 (BSA:Au = 1:1) 20 mm (BSA:Au = 1:50) hplc グレードの水。 注: 異なるギア比の BSA のゴールドは励起発光マップの大幅に異なる赤蛍光パターンになります。</li>
	</ol>
</li>
	<li>37 ° C の水浴の BSA の反応バイアルを置き、精力的に磁性攪拌器を使用して 750 rpm で攪拌します。</li>
	<li>攪拌を開始した後すぐに 1 mL の塩化金酸をソリューションに追加します。ソリューションの色は、黄色にクリアから変換する必要があります。</li>
	<li>磁性攪拌器を使用して 750 rpm、37 ° C で 2 分の混合物をかき混ぜなさい。</li>
	<li>反応バイアルに 1 M NaOH の 100 μ L を 12 の pH をもたらすにソリューションに追加します。 注: 水酸化ナトリウムを追加すると、直後にソリューションする必要があります黄色茶色に少し暗くなるし、黄色に再度オンします。</li>
	<li>2 h 750 rpm、37 ° C で攪拌し続けるソリューションは黄色から濃い黄色/茶色色にゆっくりと変更ください。この色の変化は、複雑な赤の蛍光 BSA-Au(III) の形成を示します。</li>
	<li>2 日間室温で放置し、サンプルを許可するソリューションは引き続き黄色茶色に暗くし、蛍光強度が増加します。<ol>
<li>また、ソリューションの色が黄色茶色に進化するにつれてより多くの h を 12 の 37 ° C で攪拌し続けるサンプルをしましょう。</li>
	</ol>
</li>
</ol>2. BSA-Cu(II)-Au(III) の合成
<ol><li>HPLC グレードの水 1 mL に BSA の 25 mg を溶解します。ソリューションが明確に表示されます。</li>
	<li>5 mM の濃度に HPLC グレードの水で銅の (II) 塩化物水和物を溶解します。ソリューションは、光を表示する必要があります青。</li>
	<li>5 mL の反応バイアルに BSA 溶液の 1 mL を追加し、37 ° C の水浴の配置750 rpm で混合物をかき混ぜます。</li>
	<li>すぐに反応バイアルに銅の (II) 塩化物水和物溶液 0.5 mL を加え、2 分混ぜます。ソリューションは引き続きライト ブルー。</li>
	<li>12 の pH をもたらすし、2 h のミックスすることができます 1 M NaOH の 75 μ L を追加します。ソリューションは、紫色になります。</li>
	<li>5 mM の濃度に HPLC グレードの水で塩化金酸を溶解します。</li>
	<li>反応バイアルに水溶液中の塩化金酸 0.5 mL を追加し、1 M NaOH を使用して 12 に pH を調整します。</li>
	<li>2 h の反応混合物をかき混ぜなさい。 注: ソリューションは、茶色の色に進化すべき。</li>
</ol>3. BSA-Ni(II)-Au(III) の合成
<ol><li>HPLC グレードの水 1 mL に BSA の 25 mg を溶解します。ソリューションが明確に表示されます。</li>
	<li>5 mM の濃度に HPLC グレードの水のニッケル (II) 塩化物水和物を溶解します。ソリューションは、光を表示する必要がありますグリーン。</li>
	<li>5 mL 反応バイアルに BSA 溶液の 1 mL を追加し、水浴に 37 oC. 炒め 750 rpm で混合物を置きます。</li>
	<li>すぐに反応バイアルおよび 2 分のミックスにニッケル (II) 塩化物水和物溶液 0.5 mL を追加します。 注: ソリューションのままライト グリーン。</li>
	<li>12 の pH をもたらすし、2 h のミックスすることができます 1 M NaOH の 75 μ L を追加します。 注: ソリューションは、濃い黄色になります。</li>
	<li>5 mM の濃度に HPLC グレードの水で塩化金酸を溶解します。</li>
	<li>反応バイアルに水溶液中の塩化金酸 0.5 mL を追加し、12 に pH を調整します。</li>
	<li>2 h の反応混合物をかき混ぜなさい。 注: ソリューションは、茶色の色に進化すべき。</li>
</ol>4. [Cys34-capped-BSA]-Au(III) の合成
<ol><li>N-エチルマレイミド (NEM) 2 mg をリン酸緩衝生理食塩水 (PBS、pH 7.4) 1 mL に溶かしてください。</li>
	<li>PBS NEM 溶液 1 mL に BSA の 2 mg を溶解します。</li>
	<li>5 mL の反応バイアルに溶液を移し、1 時間 500 rpm で 20 ° C でかき混ぜます。</li>
	<li>500 mL の PBS、未反応 NEM を削除する一晩マグネチックスターラーで攪拌 50 回転で 12 kDa の透析チューブを使用して、ソリューションを dialyze します。</li>
	<li>0.4 mM の濃度に PBS で塩化金酸を溶解します。 注: ソリューションは、淡い黄色になります。</li>
	<li>反応バイアルを 37 ° C の水浴に転送します。750 rpm で攪拌します。</li>
	<li>すぐに反応バイアルに塩化金酸溶液の 1 mL を追加し、2 分のミックスすることができます。</li>
	<li>12 の pH をもたらすし、2 h のミックスすることができます反応バイアルに 1 M 水酸化ナトリウムの 75 μ L を追加します。</li>
</ol>5. [all-thiol-capped-BSA]-Au(III) の合成
<ol><li>2 M 尿素と 50 mM 重炭酸アンモニウム (NH4HCO3、pH 8.0) HPLC グレードの水のソリューションを準備します。</li>
	<li>上記のソリューションと 5 mL の反応バイアルへの転送の 1 mL に BSA の 3.3 mg を溶解します。</li>
	<li>0.25 M の原液を作る tris(2-carboxyethyl) HPLC 水 1 mL で TCEP 62.5 mg を溶解してホスフィン (TCEP)。</li>
	<li>TCEP の最終濃度が 8 mM になるまで反応バイアルに TCEP の原液を追加します。</li>
	<li>50 ° C で 1 時間湯せんでソリューションを孵化させなさい磁性攪拌器を使用して 500 rpm で攪拌します。</li>
	<li>完全に部屋の温度に冷却ソリューションを許可します。</li>
	<li>HPLC グレードの水の 1 mL の NEM の 12.5 mg を溶解することにより 100 mM NEM の原液を準備します。</li>
	<li>NEM の最終濃度が 16 mM まで反応バイアルに NEM の原液を追加します。</li>
	<li>20 ° C で 2 時間を結合するソリューションを許可します。500 rpm で攪拌します。</li>
	<li>過剰な TCEP、満場一致、尿素を削除する 50 mM NH4HCO3の 500 mL で一晩マグネチックスターラーで 50 rpm で液を攪拌 12 kDa の透析の管を使用して、ソリューションを dialyze します。</li>
	<li>反応バイアルを 37 ° C で水のお風呂に移動し、750 rpm で攪拌します。</li>
	<li>0.66 mM の濃度に HPLC グレードの水で塩化金酸を溶解します。</li>
	<li>すぐに反応バイアルに塩化金酸溶液の 1 mL を追加し、2 分のミックスすることができます。</li>
	<li>解決の pH は 12 まで 1 M NaOH を追加し、2 h ミックスし続けるソリューションします。</li>
</ol>

<ol>
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PH 12 で作製した BSA-Au(III) 化合物展示発光波長λemで赤い蛍光性 = 640 nm の紫外線 (UV) 光 λexで興奮すると = 365 nm (図 1 a)。赤い蛍光性の出現は遅いプロセスであり、最大の強度を高めるため室温で数日をかかります。赤の蛍光を高速生成する高温を利用できますが、37 ° C で反応を実行する最適な結果なります。2345 ° C 以上の温度で...

Uv (紫外線) を出展 BSA Au 化合物-顕著なと興奮の赤蛍光ストークス シフト、もともと謝らによって合成されています。1一意かつ安定した赤い蛍光センシング2,3,4の5,6、7、やナノメディシン8 のイメージングなどの分野でさまざまなアプリケーションを検索できます。 ,9,10、11,12,13。この化合物は、近年14,,1516ナノサイエンスの分野で多くの研究者によって広く研究されています。BSA Au 化合物は、Au25ナノクラ スターとして解釈されています。本手法の目標して詳細にこの化合物を調べ、赤い蛍光性の起源を理解することです。提示されたアプローチで、複数の Au 結合部位の存在と蛍光、Au25ナノクラ スターの単一サイトの核に代わるものの起源を示すことができます。その他タンパク質17,18,19金 (iii) との複合体の本質的な蛍光特性を変更する方法を勉強する、同じアプローチを使用できます。
赤蛍光 BSA Au 化合物の合成には、Au (BSA:Au) の蛍光強度と励起発光マップ (EEM)20のピークの場所を最大限に BSA のモル比の狭いコントロールが必要です。それ示すことができる、バインドする錯の複数の結合部位が存在するアスパラギン フラグメント (または Asp フラグメント、BSA の N 末端の最初の 4 つのアミノ酸残基) を含む21,22。金 (iii) を調整して赤 fluorescence([Cys34-capped-BSA]-Au(III))20の機構に関与する、BSA (システイン-34) の 34番目のアミノ酸も表示されます。すべてのシステイン Cys ジスルフィド結合を切断、すべてチオール、赤い蛍光をキャッピングは作り出された ([all-thiol-capped-BSA]-Au(III))。これは赤色の蛍光を生成する錯結合部位としてシステイン Cys 二硫化物結束の必要性を示します。
タンパク質化学の技術は、ナノ科学コミュニティの BSA-Au(III) 錯体の研究に広く使用していないされています。ただし、これらの複合体、同様 BSA 錯結合部位の詳細な理解を得るための特定の側面を理解するこれらの技術を採用する価値のあること。この資料は、これらの技術のいくつかを示すものです。

<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)

示されたプロトコルの目的は BSA、構造変更によって誘発される赤い蛍光を降伏への金 (iii) バインドのプロセスを研究する (λem = 640 nm) BSA-Au(III) 複合体の。メソッドは、赤い蛍光性の出現は、BSA のコンホメーションの pH による平衡遷移と相関していることを表示するために pH を調整します。赤蛍光 BSA-Au(III) 複合体は、BSA の「A フォーム」の構造に対応する pH 9.7、以上の調整でのみ形成できます。Au のモル比に BSA を調整して錯結合のプロセスの経過を監視するためのプロトコルを説明するとします。BSA、赤色の蛍光を出すあたり金 (iii) の最小数は、7 時より小さいです。BSA に複数の金 (iii) 結合部位の存在を説明するための手順でプロトコルについて述べる。まず、銅を追加することによって (Cu(II)) またはニッケル (Ni(II)) 陽イオン錯に続いて、この方法については明らかに結合部位錯赤の蛍光体ではないです。第二に、チオール保護剤によって BSA を変更して、別の nonfluorophore 形成錯結合部位が明らかにされます。第三に、BSA の構造を変更するには、切断とキャッピングの二硫化物結束の可能な錯結合サイトを示します。BSA のコンホメーションと錯バインディングを制御する、説明されたプロトコルは、他のタンパク質と金属イオンとの相互作用を研究に一般に適用できます。

BSA-Au(III) 複合体の蛍光からそれが観測されています BSA の本質的な青色蛍光の変換 (λem = 400 nm) 赤い蛍光 (λem = 640 nm) pH 9.7 平衡については、遷移 (図 1)。BSA-Au(III) EEM Au モル比異なる BSA では図 2に示すように、このデータは、どのように異なる励起波長で同じ発光波長が得られますモル比を変更?...

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

金陽イオン (Au(III)) ウシ血清アルブミン (BSA) としてコンフォメーション依存のユニークな BSA Au 蛍光を特徴付けるのためのと同様、提示の様々 なコンフォメーションにバインディングを勉強のためのプロトコル。

Luminophore 金の結合によるウシ血清アルブミンの様々 な立体構造の形成

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 ビュー.  University of North Carolina. 金陽イオン (Au(III)) ウシ血清アルブミン (BSA) としてコンフォメーション依存のユニークな BSA Au 蛍光を特徴付けるのためのと同様、提示の様々 なコンフォメーションにバインディングを勉強のためのプロトコル。

ビデオ: 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.

アイス結合タンパク質の調査用のLabVIEWオペ新規ナノリッター浸透圧計

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

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.

超高速液体クロマトグラフィー - 質量分析（UPLC-MS）による血清中の大規模非標的メタボロームプロファイリング

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.

共有結合は、自己組織化単分子膜の手法を用いて表面上のBMP-2の結合

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.

マイクロ波支援ポリの機能化（エチレングリコール）と連鎖重合で使用するためには樹脂ペプチドおよびヒドロゲル形成

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.

短いペプチドの自己組織化によって順序付け生体分子構造の形成

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.

蛍光ベースの氷面アフィニティーによる不凍タンパク質の氷結合平面の決定

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.

油中水型エマルジョン：疎水性顔料と水溶性クロロフィル結合タンパク質を組み立てるための新しいシステム

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.

高速液体クロマトグラフィー結合フローシンチレーションにより放射性標識およびホスホイノシチドの細胞レベルの定量化

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.

サルヴィとTOF-SIMSによる水中で水和タンパク質のその場キャラクタリゼーションで

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.

バインドの動的・電気泳動光散乱による解析 DNA 磁性粒子

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

Luminophore 金の結合によるウシ血清アルブミンの様々 な立体構造の形成

Luminophore 金の結合によるウシ血清アルブミンの様々な立体構造の形成