著者は彼らの技術支援のためにアジレントに感謝したいと思います。ケイト・マーカムとラファエル・パロミノは、Aβペプチドの合成及び精製におけるそれらの初期支援のために入金され、博士Hsiau偉リーは、原稿の図1を調製する際に彼の助けのために感謝しています。

Aβ40またはAβ42ペプチドの1分取HPLC精製<ol><li> HPLC精製のための以下の緩衝液を準備します。 <ol><li>超純水1000mlにNH 4 OH（28％溶液）の1.3ミリリットルを添加することにより、緩衝液A（20mMのNH 4 OH）を調製します。 </li><li> HPLCグレードのアセトニトリルを800mLの超純水200mlの溶液に、NH 4 OH（28％溶液）の1.3ミリリットルを添加することにより緩衝液B（20mMのNH 4 OHと80％のアセトニトリル）を準備します。 </li><li>超純水100mlにNH 4 OH（28％溶液）の100μLを添加することにより、試料の溶解緩衝液（0.1％NH 4 OH）を調製します。 </li></ol></li><li> 図1に示すように、セットアップHPLC機器。 <ol><li>緩衝液Aとポリマーチューブを使用して、HPLCポンプのインレットにBをバッファ含まれている溶媒ボトルを取り付けます。ワンピースフィッティングを用いたHPLCポンプにポリマーチューブを取り付けます。のポリマーチュービングていることを確認してください各バッファは、機器の適切な入口弁に取り付けられています。脱気装置とHPLCポンプを取り付けます。 </li><li>カップル300Å8μmの25ミリメートルх300ミリメートル取カラムの入口とHPLCポンプは、ポリマーチューブを使用して（ 図1（b）は 、左端の列、物質一覧を参照してください）。 <ol><li> 1ピース手締めフィッティングで取カラムにポリマーチューブを取り付けます。ポリマーカラムは正しい方法で配向していることを確認してください。 注：分取カラムの固定相は、ポリ（スチレン - ジビニルベンゼン）粒子から構成されています。ポリマーカラムの正しい向きは、単一​​方向矢印とアウターケーシングにマークされています。 </li></ol></li><li>ポリマーチューブを使用して、二波長検出器にカラムの出口を接続し、214 nmおよび280 nmの波長検出器を設定します。 <ol><li>のセットアップ機器メソッドオプションで検出波長パラメータを変更することにより、波長を変化させます内蔵のHPLCソフトウェア。 </li></ol></li><li> 1ピース手締めフィッティングと波長検出器の入口にポリマーチューブを取り付けます。波長検出器の出力バルブにポリマーチューブを取り付けます。 1ピース手締めフィッティングを用いたHPLC検出器の出口弁にポリマーチューブを取り付けます。これは、試料採取チューブです。 注：Aβペプチドに強い発色団の欠如は、214nmがピーク収集のための主要な紫外線（UV）波長として使用することを指示します。 </li></ol></li></ol><img alt="図1" src="/files/ftp_upload/55482/55482fig1.jpg" /> 図1：アミロイドβペプチドの精製に使用したHPLC機器の実験設定。 （A）脱気装置と、214および280nmに設定された可変波長検出器を取り付けた四HPLCポンプ。アミロイドβペプチドの精製のために使用される（B）HPLCカラムから左から右へ、25×300ミリメートル2分取カラム、7.5×300ミリメートル2セミ分取カラムおよび4.6×250ミリメートル2分析カラム; （C）HPLC分析のために使用される20μLステンレススチール注入ループとマニュアルインジェクタ。 （D）分取し、セミ分取精製のために使用される10mLのステンレススチール注入ループとマニュアルインジェクタ。 <a href="http://ecsource.jove.com/files/ftp_upload/55482/55482fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <ol start="3"><li> （HPLCソフトウェアに組み込まれたセットアップ装置メソッドオプションで）溶剤時刻表のパラメータを変更することにより、精製方法を入力して、表1に示すように、精製方法を実行するためのHPLCソフトウェアをプログラムします。 HPLCソフトの「上」ボタンをクリックすることにより、HPLCポンプをオンにします。 注：ポンプは、バッファAの開始比を供給を開始し、分取を通じてBをバッファリングしますarativeカラムおよびHPLC機器。 <ol><li> 30分が完全に平衡化するためのシステムのままにしておきます。 </li></ol></li></ol><table fo:keep-together.within-page="1"><tbody><tr><td> 時間/分 </td><td> バッファーAの％ </td><td> 緩衝液B Bの％ </td><td> 流量D / mLの分-1 </td></tr><tr><td> 0 </td><td> 80 </td><td> 20 </td><td> 6 </td></tr><tr><td> 45 </td><td> 75.5 </td><td> 24.5 </td><td> 6 </td></tr><tr><td> 45.01 </td><td> 80 </td><td> 20 </td><td> 6 </td></tr><tr><td> 52.01 </td><td> 80 </td><td> 20 </td><td> 6 </td></tr><tr><td> 52.02 </td><td> 73 </td><td> 27 </td><td> 6 </td></tr><tr><td> 85 </td><td> 73 </td><td> 27 </td><td> 6 </td></tr><tr><td> 92 </td><td> 5 </td><td> 95 C </td><td> 6 </td></tr></tbody></table> &lt;強い&gt;表1：25×300ミリメートルのポリマーカラムを用いてAβ42とAβ40ペプチドの精製のための時刻表。緩衝液A - 20 mMのNH 4 OHとH 2 O; 20mMのNH 4 OHでBが80％のMeCN / 20％H 2 O; cが前のサンプルの次の注射にカラムを洗浄するために15分間走りました。 dは HPLCの計装に6 ml /分の流速を実行するために、圧力限度は200バールに低減する必要があります。 <ol start="4"><li> Aβペプチド試料の精製 注：粗ペプチドは、自動固相ペプチド合成によって得られました。 10 <ol><li>サンプル溶解バッファー4mLの粗Aβペプチドの3ミリグラムを溶解します。室温で30〜60秒間および溶解を助けるために、40キロヘルツの周波数でサンプルを超音波処理。 </li><li> 16ゲージを取り付けた5mLのプラスチック注射器を用いて、HPLCカラム上にサンプル全体を注入ステンレス製の針。サブステップ1.3に概説されているような精製方法を実行します。 注：システムは、10mLのステンレススチール注入ループ（ 図1D）を装着したマニュアルインジェクタの使用を介して行われるべき試料注入を可能にします。希望のAβペプチドは、シャープな解像ピーク（ 図2C）として72と74分の間に溶出します。 </li><li> 50 mLコニカル遠心管に試料を採取します。収集した溶離液を直接注入質量分析を通じてAβピークの身元を確認してください。 11ストア-20℃で最大12時間のための溶離液。 注：12時間よりも長い期間のためのソリューションのストレージが原因で、ペプチドの酸化のための潜在的にはお勧めできません。 </li><li>液体窒素凍結乾燥中のAβペプチドの収集アリコート/アリコートを凍結フラッシュによって精製されたペプチドを分離します。凍結乾燥して-60℃の温度での試料とPRESを凍結乾燥を行います24時間の期間のための20ミリトールのを確認してください。 </li><li> Aβペプチドの純度を決定するために、下記のように分析用HPLCプロトコルを実行します。 6ヶ月までの期間、-20℃で、それらの凍結乾燥形態で保管ペプチド。 </li></ol></li></ol>精製されたAβタンパク質の2分析HPLC分析<ol><li>サブセクション1.1に概説されるようにHPLCバッファを準備します。上記のプロトコルの。 </li><li>セットアップ手順1.2.1あたり4.6×250ミリメートル分析カラム（ 図1B、右端の列）とに取り付けられた20μLのステンレススチール注入ループ（ 図1C）とマニュアルインジェクタと、図1に示したと同様に分析用HPLC楽器。 </li><li>ステップ1.3と同様の手順に従って、 表2に示すような分析方法を実行するためのHPLCソフトウェアをプログラムします。 </li></ol><table fo:keep-together.within-page="1"><tbody><tr><td> 時間/分</td><td> バッファーAの％ </td><td> 緩衝液B Bの％ </td><td> レート/ mLの分流量-1 </td></tr><tr><td> 0 </td><td> 95 </td><td> 5 </td><td> 1 </td></tr><tr><td> 30 </td><td> 50 </td><td> 50 </td><td> 1 </td></tr></tbody></table> 表2：AβペプチドのHPLC純度分析のための時刻表。緩衝液A - 20 mMのNH 4 OHとH 2 O; 20mMのNH 4 OHでBが80％のMeCN / 20％H 2 O。 <ol start="4"><li> Aβペプチドの純度分析 <ol><li>サンプル緩衝液中のペプチドの溶解により精製ペプチドを1mg / mLの溶液を調製します。 注：バッファのレシピは、サブセクション1.1に記載されています。タンパク質濃度は、280nm（280nmの ）でのタンパク質の吸収を測定することによって決定されます。 12メートル<html>濃度を決定するために使用されるOLAR吸光係数（ε）は、ε= 1,490 DM 3モル-1 cm -1 です 。 13 </li><li> HPLCカラムには1mg / mLの（222μM）溶液20μLを注入し、ステップ2.3で設定した分析法を実行します。 注：分析のために使用されていない残りの溶液を液体窒素中で急速冷凍し、Aβペプチドを回収するために凍結乾燥することができます。凍結乾燥の詳細は、サブセクション1.4.4に記載されています。 Aβペプチドは、16及び18分（ 図2D）との間の分析カラムから溶出します。 Aβペプチドの純度を決定するためのHPLC機器に付随する組み込みの統合解析ソフトウェアを使用してください。純度は、スペクトルの個々のピークの各々を積分し、ペプチドピーク率面積を計算することによって決定されます。一般的に、&gt; 95％の精製が確認されるべきです。 </li></ol></li></ol><img alt="&lt;/ htmlの">「図2」SRC = &quot;/ファイル/ ftp_upload / 55482 / 55482fig2.jpg&quot; /&gt; 図2：Aβ42の代表的HPLCトレース。 （A）従来のC 4シリカ精製条件：緩衝液A：H 2 O 0.1％トリフルオロ酢酸（TFA）で、緩衝液B：0.1％TFAとMeCNの（アセトニトリル）、勾配：20〜27％緩衝液B 40分、続いてアイソクラティック27％の緩衝液Bにより、 （B）調製精製25×300 mm 2のポリマーカラム、条件を用いて：緩衝液A：H 2 O 20でmMのNH 4 OH、バッファーB：20mMのNH 4 OH、勾配80％のMeCN / 20％H 2 Oを：アイソクラチック27％緩衝液Bに続いて70分間にわたって20〜27％緩衝液B; 25×300 mm 2のポリマーカラムを用いて分取精製を最適化（C）は 、条件は、サブセクションプロトコル記述テキストの1.3に位置し、表1に記載されています。 （D）分析用HPLCは、4.6×250ミリメートル2ポリマーカラムは、condを使用して、itions：緩衝液A：H 2 O 20 mMのNH 4 OH、緩衝液Bで30分間にわたって20mMのNH 4 OH、グラジエント5〜50％緩衝液Bで80％のMeCN / 20％H 2 O。部品A、BおよびCについてはAβ42に対応するピークは、アスタリスクでマークされています。パートD.質量分析に示すように、Cは、&gt; 95％の純度を明らかに部分的にマークされたAβ42のピークの回収は、Aβ42のピークの同一性を決定するために使用しました。 <a href="http://ecsource.jove.com/files/ftp_upload/55482/55482fig2large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a>

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著者らは、開示することは何もありません。

AβペプチドのHPLC精製は、精製に用いられる固定相とペプチドを溶出するために選択された移動相の両方の選択に大きく依存します。集約のためのペプチドおよび高傾向の低い等電点は、疎水性タンパク質の分離のための伝統的なクロマトグラフィー条件（C4または酸性モバイル溶離液と相まってC8固定相）をレンダリング挑戦、Aβペプチドは長時間の幅広いとして溶出すると、非解決ピーク?...

アルツハイマー病は、全世界で3,500万人を超える人々に影響を与える神経変性疾患です。疾患の発症と進展に強く関与する1は 、非常に凝集しやすい、疎水性ペプチドのアミロイドベータ（Aβ）です。 2Aβしかし、アミロイドベータ42（Aβ42）は、タンパク質の最も有毒な形態の42アミノ酸の変異体であると考えられている、長さが36〜43個のアミノ酸の範囲です。 3これは容易に拡散し、特に神経毒性実体であると考えられているオリゴマー種を形成するAβ42の能力に大部分で起因します。 図4は、Aβペプチドの我々の理解を促進するためには、日常的に高純度の材料を得ることが不可欠です。微量の不純物の存在は、劇的に、ペプチドの凝集性向特性を変化させることが示されています。 5 Traditionally、例えば、Aβのような疎水性ペプチドの高速液体クロマトグラフィー（HPLC）分離は、C 4またはC 8シリカベース固定相及び酸性移動相の組み合わせの使用を介して行われてきました。 6しかし、そのような条件は、ペプチドの精製に挑戦を提示することができます。 Aβペプチド（pIが約5.5）の低い等電点7は 、酸性条件下でいることを意味する（ 図2A）ペプチド凝集が増加し、結果としてしばしば単離することが困難である広い、非分解HPLCピークが生成されます。さらに、このようなブロードなピークは、多くの場合、ペプチドの凝集特性に影響を与え、そして一般的に劇的に産生されるペプチドの量に影響を与える可能性精製のその後のラウンドを必要とし得る不純物を含有します。 ポリ（スチレン/ジビニルベンゼン）固定相、PLRP-Sは、purifの代替手段を表し、瑛疎水性ペプチド。固定相は、異なるタンパク質とメッセンジャーリボ核酸（mRNA）の多数の精製に使用されています。 8,9 PLRP-S固定相は逆相分離のための付加的なアルキルリガンドを必要とせず、そしてより重要なペプチドの脱凝集をもたらす高いpHにおいて化学的に安定です。 7ここでは、我々は、高純度のアミロイドベータ42（Aβ42）とアミロイドベータ40（Aβ40）のペプチドをもたらす仕立てHPLC精製プロトコルを報告しています。

<table><tbody><tr><td>Agilent 1260 Infinity II quarternary pump</td><td>Agilent</td><td>G7111B</td><td>http://www.agilent.com/en-us/products/liquid-chromatography/lc-pumps-vacuum-degassers/1260-infinity-ii-quaternary-pump</td></tr><tr><td>Agilent 1260 Infinity II Dual variable wavelength detector</td><td>Agilent</td><td>G7114A</td><td>http://www.agilent.com/en-us/products/liquid-chromatography/lc-detectors/1260-infinity-ii-variable-wavelength-detector</td></tr><tr><td>Agilent 1260 Infinity II Manual Injector fitted with 10 mL stainless steel sample loop</td><td>Agilent</td><td>0101-1232</td><td>http://www.agilent.com/en-us/products/liquid-chromatography/lc-injection-systems/1260-infinity-ii-manual-injector</td></tr><tr><td>Agilent 1260 Infinity II Manual Injector fitted with 20 &amp;micro;L stainless steel sample loop</td><td>Agilent</td><td>G1328C</td><td>http://www.agilent.com/en-us/products/liquid-chromatography/lc-injection-systems/1260-infinity-ii-manual-injector</td></tr><tr><td>Ring Stand Mounting Bracket</td><td>Agilent</td><td>1400-3166</td><td></td></tr><tr><td>Agilent PLRP-S 300 &amp;Aring; 5&amp;nbsp;&amp;micro;m 4.6 x 250 mm (Analytical)</td><td>Agilent</td><td>PL1512-5501</td><td>http://www.agilent.com/en-us/products/liquid-chromatography/lc-columns/biomolecule-separations/plrp-s-for-biomolecules#features</td></tr><tr><td>A&amp;beta;42 or A&amp;beta;40 peptide</td><td></td><td></td><td>Synthesized in-house using a CEM liberty automated peptide synthesizer.</td></tr><tr><td>Ammonium Hydroxide (NH&lt;sub&gt;4&lt;/sub&gt;OH, 28% solution)</td><td>Fisher Scientific</td><td>A669-500</td><td></td></tr><tr><td>Acetonitrile</td><td>Fisher Scientific</td><td>A998-4</td><td></td></tr><tr><td>HPLC grade water</td><td>Fisher Scientific</td><td>W5-4</td><td></td></tr><tr><td>Falcon 50 mL conical centrifuge tube</td><td>Fisher Scientific</td><td>14-954-49A</td><td></td></tr><tr><td>Supelco PEEK Fitting One-piece fingertight, pkg of 5 ea</td><td>Sigma-Aldrich</td><td>Z227250</td><td></td></tr><tr><td>Normject 5&amp;nbsp;cc sterile syringe</td><td>Fisher Scientific</td><td>1481729</td><td></td></tr><tr><td>16 Gauge SS Needle</td><td>Rheodyne</td><td>3725-086</td><td></td></tr></tbody></table>

a tailored hplc purification protocol that yields high-purity amyloid beta 42 and amyloid beta 40 peptides, capable of oligomer formation

Amyloidogenic peptides such as the Alzheimer's disease-implicated Amyloid beta (A&#946;), can present a significant challenge when trying to obtain high purity material. Here we present a tailored HPLC purification protocol to produce high-purity amyloid beta 42 (A&#946;42) and amyloid beta 40 (A&#946;40) peptides. We have found that the combination of commercially available hydrophobic poly(styrene/divinylbenzene) stationary phase, polymer laboratory reverse phase - styrenedivinylbenzene (PLRP-S) under high pH conditions, enables the attainment of high purity (&gt;95%) A&#946;42 in a single chromatographic run. The purification is highly reproducible and can be amended to both semi-preparative and analytical conditions depending upon the amount of material wished to be purified. The protocol can also be applied to the A&#946;40 peptide with identical success and without the need to alter the method.

72および74分（ 図2C）との間の保持時間におけるAβペプチドに鋭い解決ピークの形成PLRP-S固定相と高pHの移動相の結果の組み合わせを使用してAβ42ペプチドの精製。ピークの身元の確認は、収集した溶離液を直接注入質量分析を介して行われます。溶離剤は、最大12時間、溶液中で-20℃で保存することができます。ストレージのより長い期間は、タンパク質...

Watch this Scientific Journal Video about A Tailored HPLC Purification Protocol That Yields High-purity Amyloid Beta 42 and Amyloid Beta 40 Peptides, Capable of Oligomer Formation at JoVE.com

A Tailored HPLC Purification Protocol That Yields High-purity Amyloid Beta 42 and Amyloid Beta 40 Peptides, Capable of Oligomer Formation

ここで我々は、オリゴマー形成が可能な高純度のアミロイドベータ42（Aβ42）とアミロイドベータ40（Aβ40）のペプチドを、もたらす仕立てHPLC精製プロトコルを報告しています。アミロイドベータは、アルツハイマー病に関与する高度に凝集しやすい、疎水性ペプチドです。ペプチドのアミロイド形成性の性質は、その精製の挑戦になります。

オリゴマー形成可能な高純度のアミロイドベータ42とアミロイドベータ40ペプチドを、利回りテーラードHPLC精製プロトコル

Amyloidogenic peptides such as the Alzheimer's disease-implicated Amyloid beta (A&#946;), can present a significant challenge when trying to obtain high ...

a-tailored-hplc-purification-protocol-that-yields-high-purity-amyloid

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

11.8K ビュー.  University of California, Santa Cruz. ここで我々は、オリゴマー形成が可能な高純度のアミロイドベータ42（Aβ42）とアミロイドベータ40（Aβ40）のペプチドを、もたらす仕立てHPLC精製プロトコルを報告しています。アミロイドベータは、アルツハイマー病に関与する高度に凝集しやすい、疎水性ペプチドです。ペプチドのアミロイド形成性の性質は、その精製の挑戦になります。 

ビデオ: A Tailored HPLC Purification Protocol That Yields High-purity Amyloid Beta 42 and Amyloid Beta 40 Peptides, Capable of Oligomer Formation

Preparation of Oligomeric &beta;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). &beta;-amyloid (A&beta;), a peptide produced in high amounts in AD, is known to reduce Long-Term Potentiation (LTP), a cellular correlate of learning and memory. Indeed, LTP impairment caused by A&beta; is a useful experimental paradigm for studying synaptic dysfunctions in AD models and for screening drugs capable of mitigating or reverting such synaptic impairments. Studies have shown that A&beta; produces the LTP disruption preferentially via its oligomeric form. Here we provide a detailed protocol for impairing LTP by perfusion of oligomerized synthetic A&beta;1-42 peptide onto acute hippocampal slices. In this video, we outline a step-by-step procedure for the preparation of oligomeric A&beta;1-42. Then, we follow an individual experiment in which LTP is reduced in hippocampal slices exposed to oligomerized A&beta;1-42 compared to slices in a control experiment where no A&beta;1-42 exposure had occurred.

Preparation of Oligomeric &#946;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

One feature of Alzheimer's Disease is the elevation of A&beta;1-42 peptide. Here we provide a protocol for preparing synthetic A&beta;1-42 oligomers, which impairs hippocampal Long-Term Potentiation, a cellular correlate of memory. This procedure is useful for investigating mechanisms of A&beta;-induced pathology and drug screening.

β-アミロイドオリゴマーの調製 1から42まで</sub海馬スライスでのシナプス可塑性の減損の>と誘導

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). ...

神経科学

Selection of Aptamers for Amyloid &beta;-Protein, the Causative Agent of Alzheimer&#39;s Disease

Alzheimer's disease (AD) is a progressive, age-dependent, neurodegenerative disorder with an insidious course that renders its presymptomatic diagnosis difficult1. Definite AD diagnosis is achieved only postmortem, thus establishing presymptomatic, early diagnosis of AD is crucial for developing and administering effective therapies2,3.
Amyloid &beta;-protein (A&beta;) is central to AD pathogenesis. Soluble, oligomeric A&beta; assemblies are believed to affect neurotoxicity underlying synaptic dysfunction and neuron loss in AD4,5. Various forms of soluble A&beta; assemblies have been described, however, their interrelationships and relevance to AD etiology and pathogenesis are complex and not well understood6. Specific molecular recognition tools may unravel the relationships amongst A&beta; assemblies and facilitate detection and characterization of these assemblies early in the disease course before symptoms emerge. Molecular recognition commonly relies on antibodies. However, an alternative class of molecular recognition tools, aptamers, offers important advantages relative to antibodies7,8. Aptamers are oligonucleotides generated by in-vitro selection: systematic evolution of ligands by exponential enrichment (SELEX)9,10. SELEX is an iterative process that, similar to Darwinian evolution, allows selection, amplification, enrichment, and perpetuation of a property, e.g., avid, specific, ligand binding (aptamers) or catalytic activity (ribozymes and DNAzymes).
Despite emergence of aptamers as tools in modern biotechnology and medicine11, they have been underutilized in the amyloid field. Few RNA or ssDNA aptamers have been selected against various forms of prion proteins (PrP)12-16. An RNA aptamer generated against recombinant bovine PrP was shown to recognize bovine PrP-&beta;17, a soluble, oligomeric, &beta;-sheet-rich conformational variant of full-length PrP that forms amyloid fibrils18. Aptamers generated using monomeric and several forms of fibrillar &beta;2-microglobulin (&beta;2m) were found to bind fibrils of certain other amyloidogenic proteins besides &beta;2m fibrils19. Ylera et al. described RNA aptamers selected against immobilized monomeric A&beta;4020. Unexpectedly, these aptamers bound fibrillar A&beta;40. Altogether, these data raise several important questions. Why did aptamers selected against monomeric proteins recognize their polymeric forms? Could aptamers against monomeric and/or oligomeric forms of amyloidogenic proteins be obtained? To address these questions, we attempted to select aptamers for covalently-stabilized oligomeric A&beta;4021 generated using photo-induced cross-linking of unmodified proteins (PICUP)22,23. Similar to previous findings17,19,20, these aptamers reacted with fibrils of A&beta; and several other amyloidogenic proteins likely recognizing a potentially common amyloid structural aptatope21. Here, we present the SELEX methodology used in production of these aptamers21.

Selection of Aptamers for Amyloid &#946;-Protein, the Causative Agent of Alzheimer's Disease

Aptamers are short ribo-/deoxyribo-oligonucleotides selected by in-vitro evolution methods based on affinity for a specific target. Aptamers are molecular recognition tools with versatile therapeutic, diagnostic, and research applications. We demonstrate methods for selection of aptamers for amyloid &beta;-protein, the causative agent of Alzheimer's disease.

アミロイドβ-蛋白質、アルツハイマー病の原因物質のためのアプタマーの選択

Alzheimer's disease (AD) is a progressive, age-dependent, neurodegenerative disorder with an insidious course that renders its presymptomatic diagnosis ...

A11-positive &#946;-amyloid Oligomer Preparation and Assessment Using Dot Blotting Analysis

&#946;-amyloid (A&#946;) is a hydrophobic peptide with an intrinsic tendency to self-assemble into aggregates. Among various aggregates, A&#946; oligomer is widely accepted as the leading neurotoxin in the progress of Alzheimer&#39;s disease (AD) and is considered to be the crucial event in the pathogenesis of AD. Therefore, A&#946; oligomer inhibitors might prevent neurodegeneration and have the potential to be developed as disease-modifying treatments of AD. However, different formation protocols of A&#946; oligomer might lead to oligomers with different characteristics. Moreover, there are not many methods to effectively screen A&#946;1-42 oligomer inhibitors. An A11 antibody can react with a subset of toxic A&#946;1-42 oligomer with anti-parallel &#946;-sheet structures. In this protocol, we describe how to prepare an A11-positive A&#946;1-42 oligomer-rich sample from a synthetic A&#946;1-42 peptide in vitro and to evaluate relative amounts of A11-positive A&#946;1-42 oligomer in samples by a dot blotting analysis using A11 and A&#946;1-42-specific 6E10 antibodies. Using this protocol, inhibitors of A11-positive A&#946;1-42 oligomer can also be screened from semi-quantitative experimental results.

This protocol describes how to prepare A&#946; oligomers from a synthetic peptide in vitro and to evaluate relative amounts of A&#946; oligomer by a dot blotting analysis.

A11 肯定的なアミロイド β オリゴマーの合成と評価を使用してドットのしみが付く分析

&#946;-amyloid (A&#946;) is a hydrophobic peptide with an intrinsic tendency to self-assemble into aggregates. Among various aggregates, A&#946; oligomer ...

生化学

Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a large extracellular domain and a short cytosolic domain termed the APP intracellular domain (AICD). During maturation through the secretory pathway, APP can be cleaved by proteases termed &alpha;, &beta;, and &gamma;-secretases1. Sequential proteolytic cleavage of APP with &beta; and &gamma;-secretases leads to the production of a small proteolytic peptide, termed A&beta;, which is amyloidogenic and the core constituent of senile plaques. The AICD is also liberated from the membrane after secretase processing, and through interactions with Fe65 and Tip60, can translocate to the nucleus to participate in transcription regulation of multiple target genes2,3. Protein-protein interactions involving the AICD may affect trafficking, processing, and cellular functions of holo-APP and its C-terminal fragments. We have recently shown that AICD can aggregate in vitro, and this process is inhibited by the AD-implicated molecular chaperone ubiquilin-14. Consistent with these findings, the AICD has exposed hydrophobic domains and is intrinsically disordered in vitro5,6, however it obtains stable secondary structure when bound to Fe657. We have proposed that ubiquilin-1 prevents inappropriate inter- and intramolecular interactions of AICD, preventing aggregation in vitro and in intact cells4. While most studies focus on the role of APP in the pathogenesis of AD, the role of AICD in this process is not clear. Expression of AICD has been shown to induce apoptosis8, to modulate signaling pathways9, and to regulate calcium signaling10. Over-expression of AICD and Fe65 in a transgenic mouse model induces Alzheimer's like pathology11, and recently AICD has been detected in brain lysates by western blotting when using appropriate antigen retrieval techniques12. To facilitate structural, biochemical, and biophysical studies of the AICD, we have developed a procedure to produce recombinantly large amounts of highly pure AICD protein. We further describe a method for inducing the in vitro thermal aggregation of AICD and analysis by atomic force microscopy. The methods described are useful for biochemical, biophysical, and structural characterization of the AICD and the effects of molecular chaperones on AICD aggregation.

A method for large-scale purification of the APP intracellular domain (AICD) is described. We also describe methodology to induce in vitro AICD aggregation and visualization by atomic force microscopy. The methods described are useful for biochemical/structural characterization of the AICD and the effects of molecular chaperones on its aggregation.

アミロイド前駆体タンパク質細胞内ドメインの精製と集約

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a ...

医学

Synthesis and Characterization of 1,2-Dithiolane Modified Self-Assembling Peptides

This report focuses on the synthesis of an N-terminus 1,2-dithiolane modified self-assembling peptide and the characterization of the resulting self-assembled supramolecular structures. The synthetic route takes advantage of solid-phase peptide synthesis with the on-resin coupling of the dithiolane precursor molecule, 3-(acetylthio)-2-(acetylthiomethyl)propanoic acid, and the microwave-assisted thioacetate deprotection of the peptide N-terminus before final cleavage from the resin to yield the 1,2-dithiolane modified peptide. After the high-performance liquid chromatography (HPLC) purification of the 1,2-dithiolane peptide, derived from the nucleating core of the A&#946; peptide associated with Alzheimer&#39;s disease, the peptide is shown to self-assemble into cross-&#946; amyloid fibers. Protocols to characterize the amyloid fibers by Fourier-transform infrared spectroscopy (FT-IR), circular dichroism spectroscopy (CD) and transmission electron microscopy (TEM) are presented. The methods of N-terminal modification with a 1,2-dithiolane moiety to well-characterized self-assembling peptides can now be explored as model systems to develop post-assembly modification strategies and explore dynamic covalent chemistry on supramolecular peptide nanofiber surfaces.

A protocol for the synthesis of a 1,2-dithiolane modified peptide and the characterization of the supramolecular structures resulting from the peptide self-assembly.

1, 2-ジチオランの合成とキャラクタリゼーション自己組織化ペプチド

This report focuses on the synthesis of an N-terminus 1,2-dithiolane modified self-assembling peptide and the characterization of the resulting ...

化学

Modeling Amyloid-&#946;42 Toxicity and Neurodegeneration in Adult Zebrafish Brain

Alzheimer's disease (AD) is a debilitating neurodegenerative disease in which accumulation of toxic amyloid-&#946;42 (A&#946;42) peptides leads to synaptic degeneration, inflammation, neuronal death, and learning deficits. Humans cannot regenerate lost neurons in the case of AD in part due to impaired proliferative capacity of the neural stem/progenitor cells (NSPCs) and reduced neurogenesis. Therefore, efficient regenerative therapies should also enhance the proliferation and neurogenic capacity of NSPCs. Zebrafish (Danio rerio) is a regenerative organism, and we can learn the basic molecular programs with which we could design therapeutic approaches to tackle AD. For this reason, the generation of an AD-like model in zebrafish was necessary. Using our methodology, we can introduce synthetic derivatives of A&#946;42 peptide with tissue penetrating capability into the adult zebrafish brain, and analyze the disease pathology and the regenerative response. The advantage over the existing methods or animal models is that zebrafish can teach us how a vertebrate brain can naturally regenerate, and thus help us to treat human neurodegenerative diseases better by targeting endogenous NSPCs. Therefore, the amyloid-toxicity model established in the adult zebrafish brain may open new avenues for research in the field of neuroscience and clinical medicine. Additionally, the simple execution of this method allows for cost-effective and efficient experimental assessment. This manuscript describes the synthesis and injection of A&#946;42 peptides into zebrafish brain.

This protocol describes the synthesis, characterization, and injection of monomeric amyloid-&#946;42 peptides for generating amyloid toxicity in adult zebrafish to establish an Alzheimer&#39;s disease model, followed by histological analyses and detection of aggregations.

Β42 アミロイド毒性と大人のゼブラフィッシュ脳の神経変性をモデリング

Alzheimer's disease (AD) is a debilitating neurodegenerative disease in which accumulation of toxic amyloid-&#946;42 (A&#946;42) peptides leads to ...

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 ...

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures of polypeptides fulfill many purposes, it is now recognized that most proteins can adopt an alternative assembly of beta-sheet rich amyloid. Insoluble amyloid fibrils are initially associated with multiple human ailments, but they are increasingly shown as functional players participating in various important cellular processes. In addition, amyloid deposited in patient tissues contains nonproteinaceous components, such as nucleic acids and glycosaminoglycans (GAGs). These cofactors can facilitate the formation of amyloid, resulting in the generation of different types of insoluble precipitates. By taking advantage of our understanding how proteins misfold via an intermediate stage of soluble amyloid precursor, we have devised a method to convert native proteins to amyloid fibrils in vitro. This approach allows one to prepare amyloid in large quantities, examine the properties of amyloid generated from specific proteins, and evaluate the structural changes accompanying the conversion.

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins can either adopt a native structure or misfold into insoluble amyloid. Conditions that favor the misfolding pathway lead to the formation of different types of amyloid fibrils. The methods described here allow rapid conversion of native proteins into amyloid in vitro.

天然のタンパク質からアミロイドの迅速な生成<em&gt;インビトロ</em

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures ...

生物学

Biochemical Purification and Proteomic Characterization of Amyloid Fibril Cores from the Brain

Proteinaceous fibrillar inclusions are key pathological hallmarks of multiple neurodegenerative diseases. In the early stages of Alzheimer&#39;s disease (AD), amyloid-beta peptides form protofibrils in the extracellular space, which act as seeds that gradually grow and mature into large amyloid plaques. Despite this basic understanding, current knowledge of the amyloid fibril structure, composition, and deposition patterns in the brain is limited. One major barrier has been the inability to isolate highly purified amyloid fibrils from brain extracts. Affinity purification and laser capture microdissection-based approaches have been previously used to isolate amyloids but are limited by the small quantity of material that can be recovered. This novel, robust protocol describes the biochemical purification of amyloid plaque cores using sodium dodecyl sulfate (SDS) solubilization with sucrose density gradient ultracentrifugation and ultrasonication and yields highly pure fibrils from AD patients and AD model brain tissues. Mass spectrometry (MS)-based bottom-up proteomic analysis of the purified material represents a robust strategy to identify nearly all the primary protein components of amyloid fibrils. Previous proteomic studies of proteins in the amyloid coronae have revealed an unexpectedly large and functionally diverse collection of proteins. Notably, after refining the purification strategy, the number of co-purifying proteins was reduced by more than 10-fold, indicating the high purity of the isolated SDS insoluble material. Negative staining and immuno-gold electron microscopy allowed confirmation of the purity of these preparations. Further studies are required to understand the spatial and biological attributes that contribute to the deposition of these proteins into amyloid inclusions. Taken together, this analytical strategy is well-positioned to increase the understanding of amyloid biology.

This biochemical purification method with mass spectrometry-based proteomic analysis facilitates the robust characterization of amyloid fibril cores, which may accelerate the identification of targets for preventing Alzheimer's disease.

脳からのアミロイド線維コアの生化学的浄化とプロテオミクス特性評価

Proteinaceous fibrillar inclusions are key pathological hallmarks of multiple neurodegenerative diseases. In the early stages of Alzheimer&#39;s disease ...

Establishing an Alzheimer&#39;s Disease Model in Zebrafish Brain Through Amyloid Peptide Injection

Bhattarai, P., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/56014">Modeling Amyloid-&#946;42 Toxicity and Neurodegeneration in Adult Zebrafish Brain.</a> J. Vis. Exp. (2017).
This video demonstrates the procedure of injecting monomeric amyloid peptides into the forebrain of adult zebrafish to generate amyloid toxicity, mimicking the neuronal damage seen in Alzheimer&#39;s disease

オリゴマー形成可能な高純度のアミロイドベータ42とアミロイドベータ40ペプチドを、利回りテーラードHPLC精製プロトコル

要約

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β-アミロイドオリゴマーの調製_{1から42までと誘導}

アミロイドβ-蛋白質、アルツハイマー病の原因物質のためのアプタマーの選択

A11 肯定的なアミロイド β オリゴマーの合成と評価を使用してドットのしみが付く分析

アミロイド前駆体タンパク質細胞内ドメインの精製と集約

1, 2-ジチオランの合成とキャラクタリゼーション自己組織化ペプチド

Β42 アミロイド毒性と大人のゼブラフィッシュ脳の神経変性をモデリング

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

天然のタンパク質からアミロイドの迅速な生成

脳からのアミロイド線維コアの生化学的浄化とプロテオミクス特性評価

アミロイドペプチド注入によるゼブラフィッシュ脳のアルツハイマー病モデルの確立

オリゴマー形成可能な高純度のアミロイドベータ42とアミロイドベータ40ペプチドを、利回りテーラードHPLC精製プロトコル

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β-アミロイドオリゴマーの調製 1から42までと誘導

アミロイドβ-蛋白質、アルツハイマー病の原因物質のためのアプタマーの選択

A11 肯定的なアミロイド β オリゴマーの合成と評価を使用してドットのしみが付く分析

アミロイド前駆体タンパク質細胞内ドメインの精製と集約

1, 2-ジチオランの合成とキャラクタリゼーション自己組織化ペプチド

Β42 アミロイド毒性と大人のゼブラフィッシュ脳の神経変性をモデリング

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

天然のタンパク質からアミロイドの迅速な生成

脳からのアミロイド線維コアの生化学的浄化とプロテオミクス特性評価

アミロイドペプチド注入によるゼブラフィッシュ脳のアルツハイマー病モデルの確立

β-アミロイドオリゴマーの調製_{1から42までと誘導}