Name: luminophore formation in various conformations of bovine serum albumin by binding of gold(iii)
Uploaded: 2018-08-31T15:00:00.000+00:00
Duration: 8 min 26 s
Description: Watch this Scientific Journal Video about Luminophore Formation in Various Conformations of Bovine Serum Albumin by Binding of GoldIII at JoVE.com

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>

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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), &gt;99.0%</td><td>Sigma-Aldrich</td><td>4259</td><td></td></tr><tr><td>Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), &gt;98.0%</td><td>Sigma-Aldrich</td><td>C4706</td><td></td></tr><tr><td>Sodium hydroxide, &gt;98.0%</td><td>Sigma-Aldrich</td><td>S8045</td><td></td></tr><tr><td>Urea, 99.5%</td><td>Chem-Implex Int&#039;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

Iridium(III) Luminescent Probe for Detection of the Malarial Protein Biomarker Histidine Rich Protein-II

This work outlines the synthesis of a non-emissive, cyclometalated Ir(III) complex, Ir(ppy)2(H2O)2+ (Ir1), which elicits a rapid, long-lived phosphorescent signal when coordinated to a histidine-containing protein immobilized on the surface of a magnetic particle. Synthesis of Ir1, in high yields,is complete O/N&#160;and involves splitting of the parent cyclometalated Ir(III) chloro-bridged dimer into two equivalents of the solvated complex. To confirm specificity, several amino acids were probed for coordination activity when added to the synthesized probe, and only histidine elicited a signal response. Using BNT-II, a branched peptide mimic of the malarial biomarker Histidine Rich Protein II (pfHRP-II), the iridium probe was validated as a tool for HRP-II detection. Quenching effects were noted in the BNT-II/Ir1 titration when compared to L-Histidine/Ir1, but these were attributed to steric hindrance and triplet state quenching. Biolayer interferometry was used to determine real-time kinetics of interaction of Ir1 with BNT-II. Once the system was optimized, the limit of detection of rcHRP-II using the probe was found to be 12.8 nM in solution. When this protein was immobilized on the surface of a 50 &#181;m magnetic agarose particle, the limit of detection was 14.5 nM. The robust signal response of this inorganic probe, as well as its flexibility of use in solution or immobilized on a surface, can lend itself toward a variety of applications, from diagnostic use to imaging.

IridiumIII Luminescent Probe for Detection of the Malarial Protein Biomarker Histidine Rich Protein-II

Robust detection reagents are of increasing necessity for developing new malaria diagnostic tools. An iridium(III) probe was designed that emits long-lasting luminescent signal in the presence of a histidine-rich malarial protein biomarker. Detection of the protein either in solution or immobilized on a magnetic particle affords flexibility in application.

マラリアのタンパク質バイオマーカーヒスチジンリッチプロテイン-IIの検出のためのイリジウム（III）発光プローブ

This work outlines the synthesis of a non-emissive, cyclometalated Ir(III) complex, Ir(ppy)2(H2O)2+ (Ir1), which elicits a rapid, long-lived ...

化学

Au-Interaction of Slp1 Polymers and Monolayer from Lysinibacillus sphaericus JG-B53 - QCM-D, ICP-MS and AFM as Tools for Biomolecule-metal Studies

In this publication the gold sorption behavior of surface layer (S-layer) proteins (Slp1) of Lysinibacillus sphaericus JG-B53 is described. These biomolecules arrange in paracrystalline two-dimensional arrays on surfaces, bind metals, and are thus interesting for several biotechnical applications, such as biosorptive materials for the removal or recovery of different elements from the environment and industrial processes. The deposition of Au(0) nanoparticles on S-layers, either by S-layer directed synthesis 1 or adsorption of nanoparticles, opens new possibilities for diverse sensory applications. Although numerous studies have described the biosorptive properties of S-layers 2-5, a deeper understanding of protein-protein and protein-metal interaction still remains challenging. In the following study, inductively coupled mass spectrometry (ICP-MS) was used for the detection of metal sorption by suspended S-layers. This was correlated to measurements of quartz crystal microbalance with dissipation monitoring (QCM-D), which allows the online detection of proteinaceous monolayer formation and metal deposition, and thus, a more detailed understanding on metal binding.
The ICP-MS results indicated that the binding of Au(III) to the suspended S-layer polymers is pH dependent. The maximum binding of Au(III) was obtained at pH 4.0. The QCM-D investigations enabled the detection of Au(III) sorption as well as the deposition of Au(0)-NPs in real-time during the in situ experiments. Further, this method allowed studying the influence of metal binding on the protein lattice stability of Slp1. Structural properties and protein layer stability could be visualized directly after QCM-D experiment using atomic force microscopy (AFM). In conclusion, the combination of these different methods provides a deeper understanding of metal binding by bacterial S-layer proteins in suspension or as monolayers on either bacterial cells or recrystallized surfaces.

Au-Interaction of Slp1 Polymers and Monolayer from Lysinibacillus sphaericus JG-B53 - QCM-D, ICP-MS and AFM as Tools for Biomolecule-metal Studies

To obtain basic information on the sorption and recycling of gold from aqueous systems the interaction of Au(III) and Au(0) nanoparticles on S-layer proteins were investigated. The sorption of protein polymers was investigated by ICP-MS and that of proteinaceous monolayers by QCM-D. Subsequent AFM enables the imaging of the nanostructures.

SLP1ポリマーと単層のAuのインタラクションから<em&gt; Lysinibacillusスフェリカス</em&gt; JG-B53 - 生体分子金属研究のためのツールと​​して、QCM-D、ICP-MSおよびAFM

In this publication the gold sorption behavior of surface layer (S-layer) proteins (Slp1) of Lysinibacillus sphaericus JG-B53 is described. These ...

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a fluorescent dye and are spotted by a photodetector following their separation. Single molecule fluorescence imaging can provide ultrasensitive protein detection with its ability for visualizing individual fluorescent molecules. However, the application of this powerful imaging method to electrophoresis assays is hampered by the lack of ways to characterize the homogeneity of fluorescent labeling of each protein species across the proteome. Here, we developed a method to evaluate the labeling homogeneity across the proteome based on a single molecule fluorescence imaging assay. In our measurement using a HeLa cell sample, the proportion of proteins labeled with at least one dye, which we termed &#8216;labeling occupancy&#8217; (LO), was determined to range from 50% to 90%, supporting the high potential of the application of single molecule imaging to sensitive and precise proteome analysis.

Here, we present a protocol to assess the labeling homogeneity for each protein species in a complex protein sample at the single molecule level.

プロテオーム的分類の単一分子レベルでの均一性の定量化

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

生化学

A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold nanoclusters (Au NCs) within the drug-loaded protein, based on the differential fluorescence signal emitted by the Au NCs. Albumin proteins such as human serum albumin (HSA) and bovine serum albumin (BSA) are selected as the model proteins. Four small molecular drugs (e.g., ibuprofen, warfarin, phenytoin, and sulfanilamide) of different binding affinities to the albumin proteins are tested. It was found that the formation rate of fluorescent Au NCs inside the drug loaded albumin protein under denaturing conditions (i.e., 60 &#176;C or in the presence of urea) is slower than that formed in the pristine protein (without drugs). Moreover, the fluorescent intensity of the as-formed NCs is found to be inversely correlated to the binding affinities of these drugs to the albumin proteins. Particularly, the higher the drug-protein binding affinity, the slower the rate of Au NCs formation, and thus a lower fluorescence intensity of the resultant Au NCs is observed. The fluorescence intensity of the resultant Au NCs therefore provides a simple measure of the relative binding strength of different drugs tested. This method is also extendable to measure the specific drug-protein binding constant (KD) by simply varying the drug content preloaded in the protein at a fixed protein concentration. The measured results match well with the values obtained using other prestige but more complicated methods.

A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

タンパク質 - 小分子標的薬のスクリーニングのための迅速かつ定量的蛍光法

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold ...

生物工学

Determining Total Protein and Bioactive Protein Concentrations in Bovine Colostrum

Colostrum is a complex biological fluid produced by mammals immediately after parturition. It meets all the nutritional requirements for neonates as a good source of macro- and micronutrients, bioactive peptides, and growth factors. Bovine colostrum is also a potential source of nutrition and bioactive because of its rich protein content that includes immunoglobulin G (IgG) and lactoferrin. However, the level of lactoferrin and IgG in bovine colostrum changes markedly during the lactation period. Therefore, monitoring the concentration of IgG and lactoferrin for the use of bovine colostrum as a protein source is an important question to study. Methods in this article describe how to determine protein content, as well as specific concentrations of lactoferrin and IgG. These methods include the following steps: Isolation of bovine colostrum proteins, Determination of protein concentration via Bicinchoninic acid assay (BCA), Visualization of proteins via SDS-PAGE, Determination of lactoferrin, and IgG concentration using an ELISA Assay.

Bovine colostrum is both a primary source of nutrients and immunological support for the newborn calf. The understanding of the level of therapeutic proteins (lactoferrin and IgG) is important for the bovine colostrum dosing and standardization for human consumption.

Colostrum is a complex biological fluid produced by mammals immediately after parturition. It meets all the nutritional requirements for neonates as a ...

Limited Proteolysis and Gel Electrophoresis in the Presence of Metal Cations: Au(III)-binding Luminescent Domain in Serum Albumins

The purpose of the presented protocols is to determine the domain of Au(III) binding in BSA. The BSA-Au(III) compound exhibits ultraviolet (UV)-excitable red luminescence (&#955;em = 640 nm), with unusual Stokes shifts compared to the innate UV/blue fluorescence arising from the aromatic residues. Red-luminescent complexes are formed in highly alkaline conditions above pH 10 and require a conformation change within the protein to occur. In addition, preservation of Cys-Cys disulfide bonds in BSA is necessary to obtain this red luminescence. In order to understand the mechanism of this luminescence, elucidation of the luminophore-forming Au(III) binding site is essential. A facile way to assess the luminophore-forming site would be to (1) predictably fragment the protein by enzymatic digestion, (2) react the obtained fragments with Au(III), then (3) perform gel electrophoresis to observe the well-separated fragment bands and analyze the in-gel red luminescence. However, due to the alkaline conditions and the reaction with metal cations, new limited proteolysis techniques and gel electrophoresis conditions must be applied. Particularly, the presence of metal cations in gel electrophoresis can make the band separations technically difficult. We describe this new protocol in steps to identify the red-luminophore-forming metal binding domain in BSA. This protocol can thus be applied for analyzing protein fragments that must remain in a non-denatured or a partially denatured state, in the presence of metal cations. Because the majority of proteins need metal cations to function, analyses of metal-bound proteins are often desired, which have relied on x-ray crystallography in the literature. This method, on the other hand, could be used in supplement to study the interactions of proteins with metal cations without requiring the protein crystallization and at a desired pH condition.

Limited Proteolysis and Gel Electrophoresis in the Presence of Metal Cations: AuIII-binding Luminescent Domain in Serum Albumins

We present a protocol for studying the binding domain of Au(III) in bovine serum albumin (BSA).

The purpose of the presented protocols is to determine the domain of Au(III) binding in BSA. The BSA-Au(III) compound exhibits ultraviolet (UV)-excitable ...

Synthesis of Near-Infrared Emitting Gold Nanoclusters for Biological Applications

Over the past decade, fluorescent gold nanoclusters (AuNCs) have witnessed growing popularity in biological applications and enormous efforts have been devoted to their development. In this protocol, a recently developed, facile method for preparation of water soluble, biocompatible, and colloidally stable near-infrared emitting AuNCs have been described in detail. This room-temperature, bottom-up chemical synthesis provides easily functionalizable AuNCs capped with thioctic acid and thiol-modified polyethylene glycol in aqueous solution. The synthetic approach requires neither organic solvents or additional ligand exchange nor extensive knowledge of synthetic chemistry to reproduce. The resulting AuNCs offer free surface carboxylic acids, which can be functionalized with various biological molecules bearing a free amine group without adversely affecting the photoluminescent properties of the AuNCs. A quick, reliable procedure for flow cytometric quantification and confocal microscopic imaging of AuNC uptake by HeLa cells also been described. Due to the large Stokes shift, proper setting of filters in flow cytometry and confocal microscopy is necessary for efficient detection of near-infrared photoluminescence of AuNCs.

A reliable and easily reproducible method for preparation of functionalizable, near-infrared emitting photoluminescent gold nanoclusters and their direct detection inside HeLa cells by flow cytometry and confocal laser scanning microscopy is described.

近赤外線発光金ナノクラスターの生物応用のための合成

Over the past decade, fluorescent gold nanoclusters (AuNCs) have witnessed growing popularity in biological applications and enormous efforts have been ...

Characterization of pH-Dependent Reversible Self-Assembly of Amyloid Beta 1-40-Coated Gold Colloids

The characterization of A&#946;1-40 coated over the nano-gold colloidal particle surfaces was conducted using surface plasmon resonance (SPR) spectroscopy and transmission electron microscopy (TEM). The observed pH-dependent shift of the SPR band of A&#946;1-40-coated 20 nm gold particles was correlated with alternation of aggregation and disaggregation observed in the TEM images. A pH of ~4&#160;induced an unfolded conformation, and a pH of ~10&#160;induced a folded conformation of A&#946;1-40 on the gold surface. This reversible aggregation process was observed by Raman imaging as the pH was gradually changed from pH 4 to pH 10. We observed a pH-dependent morphology change in A&#946;1-40 on the gold surface, where clear aggregates were observed at pH 4. However, we observed very subtle differences in the surface-enhanced Raman spectroscopy (SERS) spectrum between pH 4 and pH 10 conditions, with the most striking difference being spectral density in the region of 250 cm-1 and 1750 cm-1. Specifically, the mode analysis of the reversible aggregation indicated that the aggregates were formed by the unfolded conformation of A&#946;1-40 which involved the benzene ring section of Tyrosine and Phenylalanine. Conversely, the disassembly of the aggregates was associated with conformational changes in protein folding of A&#946;1-40 involving Histidine, Glutamine, Methionine, and Aspartic acid.

Here, we present a systematic method describing the quasi-reversible self-assembly of the amyloid beta 1-40 (A&#946;1-40)-coated 20 nm gold aggregates. The nano-size-dependent quasi-reversible networking between peptides was correlated with specific amino acids or sections of the A&#946;1-40 monomer.

アミロイドβ 1-40被覆金コロイドのpH依存性可逆的自己組織化の特性評価

The characterization of A&#946;1-40 coated over the nano-gold colloidal particle surfaces was conducted using surface plasmon resonance (SPR) spectroscopy ...

Real-time Monitoring of Ligand-receptor Interactions with Fluorescence Resonance Energy Transfer

FRET is a process whereby energy is non-radiatively transferred from an excited donor molecule to a ground-state acceptor molecule through long-range dipole-dipole interactions1. In the present sensing assay, we utilize an interesting property of PDA: blue-shift in the UV-Vis electronic absorption spectrum of PDA (Figure 1) after an analyte interacts with receptors attached to PDA2,3,4,7. This shift in the PDA absorption spectrum provides changes in the spectral overlap (J) between PDA (acceptor) and rhodamine (donor) that leads to changes in the FRET efficiency. Thus, the interactions between analyte (ligand) and receptors are detected through FRET between donor fluorophores and PDA. In particular, we show the sensing of a model protein molecule streptavidin. We also demonstrate the covalent-binding of bovine serum albumin (BSA) to the liposome surface with FRET mechanism. These interactions between the bilayer liposomes and protein molecules can be sensed in real-time. The proposed method is a general method for sensing small chemical and large biochemical molecules. Since fluorescence is intrinsically more sensitive than colorimetry, the detection limit of the assay can be in sub-nanomolar range or lower8. Further, PDA can act as a universal acceptor in FRET, which means that multiple sensors can be developed with PDA (acceptor) functionalized with donors and different receptors attached on the surface of PDA liposomes.

We demonstrate FRET between conjugated polymer polydiacetylene (PDA) and fluorophore attached to the surface of PDA liposomes for the sensing of biomolecules. PDA liposomes also contained receptor molecules on their surfaces for biomolecules to be used as probes. Ligand-receptor interactions lead to changes in the FRET efficiency between the fluorophore and PDA which is the basis of the sensing mechanism.

蛍光共鳴エネルギー移動によるリガンド - 受容体相互作用のリアルタイム監視

FRET is a process  whereby energy is non-radiatively transferred from an excited donor molecule to  a ground-state acceptor molecule through long-range ...

生物学

Development of a Colloidal Gold-based Immunochromatographic Test Strip for Detection of Cetacean Myoglobin

This protocol describes the development of a colloidal gold immunochromatographic test strip based on the sandwich format that can be used to differentiate the myoglobin (Mb) of cetaceans from that of seals and other animals. The strip provides rapid and on-the-spot screening for cetacean meat, thereby restraining its illegal trade and consumption. Two monoclonal antibodies (mAbs) with reactivity toward the Mb of cetaceans were developed. The amino acid sequences of Mb antigenic reactive regions from various animals were analyzed in order to design two synthetic peptides (a general peptide and a specific peptide) and thereafter raise the mAbs (subclass IgG1). The mAbs were selected from hybridomas screened by indirect ELISA, western blot and dot blot. CGF5H9 was specific to the Mbs of rabbits, dogs, pigs, cows, goats, and cetaceans while it showed weak to no affinity to the Mbs of chickens, tuna and seals. CSF1H13 can bind seals and cetaceans with strong affinity but showed no affinity to other animals. Cetacean samples from four families (Balaenopteridae, Delphinidae, Phocoenidae and Kogiidae) were used in this study, and the results indicated that these two mAbs have broad binding ability to Mbs from different cetaceans. These mAbs were applied on a sandwich-type colloidal gold immunochromatographic test strip. CGF5H9, which recognizes many species, was colloid gold-labeled and used as the detection antibody. CSF1H13, which was coated on the test zone, detected the presence of cetacean and seal Mbs. Muscle samples from tuna, chicken, seal, five species of terrestrial mammals and 15 species of cetaceans were tested in triplicate. All cetacean samples showed positive results and all the other samples showed negative results.

This protocol describes the development of two IgG class monoclonal antibodies (mAbs) strongly reactive to myoglobin of cetaceans. These mAbs are applied on a colloidal gold immunochromatographic test strip based on the sandwich format to differentiate the Mb of cetaceans from seal and other animals.

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

要約

さらに動画を探す

この動画の章

マラリアのタンパク質バイオマーカーヒスチジンリッチプロテイン-IIの検出のためのイリジウム（III）発光プローブ

SLP1ポリマーと単層のAuのインタラクションから

プロテオーム的分類の単一分子レベルでの均一性の定量化

タンパク質 - 小分子標的薬のスクリーニングのための迅速かつ定量的蛍光法

Determining Total Protein and Bioactive Protein Concentrations in Bovine Colostrum

Limited Proteolysis and Gel Electrophoresis in the Presence of Metal Cations: Au(III)-binding Luminescent Domain in Serum Albumins

近赤外線発光金ナノクラスターの生物応用のための合成

アミロイドβ 1-40被覆金コロイドのpH依存性可逆的自己組織化の特性評価

蛍光共鳴エネルギー移動によるリガンド - 受容体相互作用のリアルタイム監視

クジラミオグロビンの検出のための金コロイドベースのイムノクロマト法テストストリップの開発

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

要約

さらに動画を探す

この動画の章

マラリアのタンパク質バイオマーカーヒスチジンリッチプロテイン-IIの検出のためのイリジウム（III）発光プローブ

SLP1ポリマーと単層のAuのインタラクションから

プロテオーム的分類の単一分子レベルでの均一性の定量化

タンパク質 - 小分子標的薬のスクリーニングのための迅速かつ定量的蛍光法

Determining Total Protein and Bioactive Protein Concentrations in Bovine Colostrum

Limited Proteolysis and Gel Electrophoresis in the Presence of Metal Cations: Au(III)-binding Luminescent Domain in Serum Albumins

近赤外線発光金ナノクラスターの生物応用のための合成

アミロイドβ 1-40被覆金コロイドのpH依存性可逆的自己組織化の特性評価

蛍光共鳴エネルギー移動によるリガンド - 受容体相互作用のリアルタイム監視

クジラミオグロビンの検出のための金コロイドベースのイムノクロマト法テストストリップの開発

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