The authors would like to thank Agilent for their technical assistance. Kate Markham and Rafael Palomino are credited for their initial help in the synthesis and purification of the A&#946; peptide and Dr Hsiau-Wei Lee is thanked for his help in preparing Figure 1 of the manuscript.

1. Präparative HPLC-Reinigung des Aβ40 oder Aß42-Peptid <ol><li> Bereiten Sie die folgenden Puffer für die HPLC - Reinigung. <ol><li> Bereiten Puffer A (20 mM NH 4 OH) durch Zugabe von 1,3 ml NH 4 OH (28% ige Lösung) auf 1000 ml Reinstwasser. </li><li> Bereiten Puffer B (80% Acetonitril mit 20 mM NH 4 OH) durch Zugabe von 1,3 ml NH 4 OH (28% ige Lösung) zu einer Lösung von 800 ml HPLC-reines Acetonitril und 200 ml Reinstwasser. </li><li> Präparieren Probenlösungspuffer (0,1% NH 4 OH) durch Zugabe von 100 &amp; mgr; l NH 4 OH (28% ige Lösung) zu 100 ml Reinstwasser. </li></ol></li><li> Setup das HPLC - Gerät wie in Abbildung 1 dargestellt. <ol><li> Bringen Sie die Lösungsmittelflaschen, die A enthalten Puffer und Puffer B zu den Einlässen der HPLC-Pumpe mit Polymerschlauch. Bringen Sie den Polymerschlauch an die HPLC-Pumpe mit einem einteiligen Armatur. Stellen Sie sicher, dass der Polymerschlauch ausjeder Puffer an den richtigen Einlaßventil des Instruments angebracht. Den HPLC-Pumpe mit einem Entgaser. </li><li> Koppeln der HPLC - Pumpe mit dem Einlass der 300 Å 8 um 25 mm х 300 mm präparativen Säule (1B, Spalte ganz links, siehe Materialliste) polymer Schlauch verwendet wird . <ol><li> Bringen Sie den Polymerschlauch zur präparativen Säule mit einem einteiligen Finger eng anliegende. Sicherzustellen, dass die Polymersäule in der richtigen Weise ausgerichtet ist. HINWEIS: Die stationäre Phase der präparativen Säule aus Poly (styrol-divinylbenzol) -Teilchen umfassen. Die korrekte Orientierung der Polymer Säule wird mit einem einzigen Richtungspfeil auf dem äußeren Gehäuse markiert. </li></ol></li><li> Verbinden Sie den Ausgang der Säule mit dem Dual-Wellenlängen-Detektor Polymerschlauch mit und stellen Sie den Wellenlängen-Detektor auf 214 nm und 280 nm. <ol><li> Ändern Sie die Wellenlänge, die durch die Detektionswellenlängenparameter im Setup-Instrument Methode Option der VeränderungEinbau-HPLC-Software. </li></ol></li><li> Bringen Sie den Polymerschlauch mit dem Einlass des Wellenlängendetektor mit einem einteiligen Finger eng anliegende. Befestigen Polymerschlauch mit dem Ausgangsventil des Wellenlängendetektors. Bringen Sie den Polymerschlauch mit dem Auslassventil des HPLC-Detektor mit einem einteiligen Finger eng anliegende. Dies wird die Probensammelschlauch sein. HINWEIS: Das Fehlen eines starken Chromophor auf das Aß-Peptid diktiert, dass 214 nm für die Peaksammlung als primäre ultravioletter (UV) Wellenlängen verwendet werden. </li></ol></li></ol><img alt="Abbildung 1" src="/files/ftp_upload/55482/55482fig1.jpg" /> Abbildung 1: Experimenteller Aufbau des HPLC Instrument zur Reinigung der amyloiden beta - Peptiden verwendet. (A) Die Pumpe quartären HPLC , ausgestattet mit einem Entgaser und Detektor mit variabler Wellenlänge auf 214 nm und 280 nm eingestellt; (B) HPLC - Säulen zur Reinigung der Amyloid - beta - Peptide verwendet, vonvon links nach rechts, 25 x 300 mm 2 präparativen Säule, 7,5 x 300 mm 2 semi präparativen Säule und 4,6 x 250 mm 2 analytische Säule; (C) Hand Injektor mit 20 &amp; mgr; l Edelstahl - Injektionsschleife für analytische HPLC verwendet wird ; (D) Manueller Injektor mit 10 ml Edelstahl - Injektionsschleife für die präparative und semi präparative Reinigung verwendet. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55482/55482fig1large.jpg" target="_blank">Bitte klicken Sie hier</a> , um <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55482/55482fig1large.jpg" target="_blank">eine größere Version dieser Figur zu sehen.</a> <ol start="3"><li> Programmieren der HPLC - Software die Reinigungsverfahren , wie gezeigt in Tabelle 1. Geben Sie den Reinigungsverfahren durch Ändern des Lösungsmittelfahrplanparameter (im Setup - Instrument Verfahren Option eingebaut in die HPLC - Software) ausgeführt werden . Schalten Sie die HPLC - Pumpe durch die &quot;on&quot; Taste auf der HPLC - Software klicken. HINWEIS: Die Pumpe Ausgangsverhältnis von Puffer A Versorgung beginnt und Puffer B durch die preparative Säule und die HPLC-Instrument. <ol><li> Verlassen das System für 30 min vollständig äquilibrieren. </li></ol></li></ol><table fo:keep-together.within-page="1"><tbody><tr><td> Zeit / min </td><td> % Puffer A a </td><td> % Puffer B b </td><td> Durchflussrate d / mL min -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;strong&gt; Tabelle 1: Zeitplan für die Reinigung der Aß42 und Aβ40 Peptide, die die Säule 25 × 300 mm Polymer verwendet wird. einen Puffer A - H 2 O mit 20 mM NH 4 OH; b 80% MeCN / 20% H 2 O mit 20 mM NH 4 OH; c lief für 15 min , um die Säule vor der nächsten Injektion von Probe zu waschen; d Um eine Strömungsgeschwindigkeit von 6 ml / min auf die HPLC - Instrumentierung zu laufen, muss die Druckgrenze auf 200 bar reduziert werden. <ol start="4"><li> Reinigung des Aß - Peptidprobe Anmerkung: Das Rohpeptid durch automatisierte Festphasen-Peptidsynthese erhalten wurde. 10 <ol><li> Man löst 3 mg rohes Aß Peptid in 4 ml der Probenlösungspuffer. Beschallen die Probe für 30-60 s bei Raumtemperatur und bei einer Frequenz von 40 kHz Auflösung zu unterstützen. </li><li> Injizieren Sie die gesamte Probe auf die HPLC-Säule eine 5 ml Plastikspritze mit einer 16-Gauge ausgestattet mitNadel aus rostfreiem Stahl. Führen Sie das Reinigungsverfahren wie in Teilschritt 1.3 skizziert. HINWEIS: Das System ermöglicht eine Probeninjektion durch den Einsatz einer manuellen Injektor mit einer 10 ml Edelstahl - Injektionsschleife (1D) angebracht erfolgen. Die gewünschte Aß - Peptid eluieren zwischen 72 und 74 min als scharfe gelöst Peak (2C). </li><li> Sammeln der Probe in einem 50 ml konischen Zentrifugenröhrchen. Bestätigen Sie die Identität des Aß-Spitze durch direkte Injektion Massenspektrometrie des gesammelten Eluenten. 11 Bewahren Sie das Eluens für bis zu 12 Stunden bei -20 ° C. HINWEIS: Die Lagerung der Lösung für einen Zeitraum länger als 12 h nicht aufgrund zur Oxidation des Peptids auf das Potential empfohlen. </li><li> Isolieren Sie das gereinigte Peptid durch Flash Einfrieren des gesammelten aliquoten / Aliquots des Aß-Peptid in flüssigem Stickstoff und lyophilisieren. Führen Lyophilisation durch Gefriertrocknung der Probe bei einer Temperatur von -60 ° C und einem pressicher von 20 mTorr für einen Zeitraum von 24 h. </li><li> Führen Sie die analytische HPLC-Protokolls, wie unten beschrieben, die Reinheit des Aß-Peptids zu bestimmen. Store-Peptide in ihrer lyophilisierten Form bei -20 ° C für einen Zeitraum von bis zu 6 Monaten. </li></ol></li></ol> 2. Analytische HPLC-Analyse des gereinigten Aß-Protein <ol><li> Bereiten Sie die HPLC-Puffer, wie in Unterabschnitt 1.1 beschrieben. des obigen Protokolls. </li><li> Setup die analytische HPLC gemäß Schritt 1.2.1 und wie in Figur 1 mit dem 4,6 × 250 mm analytische Säule (1B, Spalte ganz rechts) und dem manuellen Injektor mit 20 &amp; mgr; l Edelstahl - Injektionsschleife dargestellt (Abbildung 1C) ausgerüstet der Instrument. </li><li> Programmieren der HPLC - Software die analytischen Verfahren , wie in Tabelle 2 gezeigt folgenden Anweisungen ähnlich zu denen in Schritt 1.3 ausgeführt werden . </li></ol><table fo:keep-together.within-page="1"><tbody><tr><td> Zeit /min </td><td> % Puffer A a </td><td> % Puffer B b </td><td> Durchflussrate / ml min -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> Tabelle 2: Zeitplan für die HPLC - Reinheitsanalyse des Aß - Peptid. einen Puffer A - H 2 O mit 20 mM NH 4 OH; b 80% MeCN / 20% H 2 O mit 20 mM NH 4 OH. <ol start="4"><li> Reinheitsanalyse des Aß - Peptids <ol><li> Bereiten einer 1 mg / ml-Lösung des gereinigten Peptids durch Auflösen des Peptides in der Probenpufferlösung. HINWEIS: Die Puffer Rezept kann in Unterabschnitt 1.1. Proteinkonzentration wird durch Messung der Protein - Absorption bei 280 nm (A 280 nm) bestimmt. 12 Die m<html> olar Extinktionskoeffizienten (ε) verwendet Konzentration zu bestimmen ist ε = 1.490 dm 3 mol -1 cm -1. 13 </li><li> Injizieren Sie 20 ul der 1 mg / ml (222 &amp; mgr; M) Lösung auf die HPLC-Säule und führen Sie die analytische Methode, die Einrichtung in 2,3 Schritt war. HINWEIS: Die verbleibende Lösung nicht für die Analyse verwendet werden, können in flüssigem Stickstoff eingefroren und lyophilisiert, um das Aß-Peptid zu gewinnen. Die Gefriertrocknung Details können in Unterabschnitt 1.4.4 zu finden. Die Aß - Peptid werden aus der analytischen Säule zwischen 16 und 18 min (2D) eluieren. Verwenden Sie die integrierte Integrationsanalyse-Software, die die HPLC-Instrument begleitet die Reinheit des Aß-Peptids zu bestimmen. Die Reinheit wird durch die Integration von jedem der einzelnen Peaks im Spektrum bestimmt und Berechnen Peptid-peak Prozentbereich. Typischerweise sollte eine Reinigung von&gt; 95% festgestellt werden. </li></ol></li></ol><img alt="&lt;/ Html">&quot;2&quot; src = &quot;/ files / ftp_upload / 55482 / 55482fig2.jpg&quot; /&gt; Abbildung 2: Repräsentative HPLC Spuren von Aß42. (A) Traditionelle C 4 Silika Reinigung Bedingungen: Puffer A: H 2 O mit 0,1% Trifluoressigsäure (TFA), Puffer B: MeCN (Acetonitril) mit 0,1% TFA, Gradient: 20 bis 27% Puffer B über 40 min , gefolgt durch isokratische 27% Puffer B; (B) Die präparative Reinigung der 25 x 300 mm 2 Polymer Spalte, Bedingungen: Puffer A: H 2 O mit 20 mM NH 4 OH, Puffer B: 80% MeCN / 20% H 2 O mit 20 mM NH 4 OH, Gradient : 20 bis 27% Puffer B über 70 min durch isokratische 27% Puffer B; (C) Optimierte präparative Reinigung der 25 x 300 mm 2 Polymer Spalte werden die Bedingungen in Tabelle 1 beschrieben , befindet sich in Unterabschnitt 1.3 des Protokolls Beschreibungstext; (D) unter Verwendung Analytische HPLC die 4,6 x 250 mm 2 Polymer - Säule, Ltgitions: Puffer A: H 2 O mit 20 mM NH 4 OH, Puffer B: 80% MeCN / 20% H 2 O mit 20 mM NH 4 OH, Gradienten-5 bis 50% Puffer B über 30 min. Für Teile A, B und C der Spitze-Aß42 entspricht, wird durch ein Sternchen gekennzeichnet. Sammlung des markierten Aß42 peak in Teil C mit einer Reinheit von&gt; 95% ergibt, wie in Teil D. Massenspektrometrie gezeigt wurde verwendet, um die Identität des Aß42 Peak zu bestimmen. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55482/55482fig2large.jpg" target="_blank">Bitte klicken Sie hier</a> , um <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55482/55482fig2large.jpg" target="_blank">eine größere Version dieser Figur zu sehen.</a>

<ol>
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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+Enhance+the+Aggregation+Propensity+of+the+Alzheimer's+A&#946;+Peptide.">Mutations Enhance the Aggregation Propensity of the Alzheimer's A&#946; Peptide.</a> J Mol Bio. 377 (2), 565-574 (2008).</li><li>Fezoui, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+method+of+preparing+the+amyloid+beta-protein+for+fibrillogenesis+and+neurotoxicity+experiments.">An improved method of preparing the amyloid beta-protein for fibrillogenesis and neurotoxicity experiments.</a> Amyloid. 7 (3), 166-178 (2000).</li><li>Zhelev, N. Z., Barratt, M. J., Mahadevan, L. 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Mass Spectrom. 13 (23), 2348-2351 (1999).</li><li>Layne, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectrophotometric+and+turbidimetric+methods+for+measuring+proteins.">Spectrophotometric and turbidimetric methods for measuring proteins.</a> Met. Enzymology. 3, 447-455 (1957).</li><li>Ioannou, J. C., Donald, A. M., Tromp, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+the+secondary+structure+changes+occurring+in+high+density+systems+of+BLG+dissolved+in+aqueous+pH+3+buffer.">Characterizing the secondary structure changes occurring in high density systems of BLG dissolved in aqueous pH 3 buffer.</a> Food Hydro. 46, 216-225 (2015).</li><li>Rahimi, F., Maiti, P., Bitan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photo-Induced+Cross-Linking+of+Unmodified+Proteins+(PICUP)+Applied+to+Amyloidogenic+Peptides.">Photo-Induced Cross-Linking of Unmodified Proteins (PICUP) Applied to Amyloidogenic Peptides.</a> J. Vis. Exp. (23), e1071 (2009).</li><li>Bitan, G., Kirkitadze, M. D., Lomakin, A., Vollers, S. S., Benedek, G. B., Teplow, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+&#946;-protein+(A&#946;)+assembly:+A&#946;40+and+A&#946;42+oligomerize+through+distinct+pathways.">Amyloid &#946;-protein (A&#946;) assembly: A&#946;40 and A&#946;42 oligomerize through distinct pathways.</a> Proc. Natl. Acad. Sci. USA. 100 (1), 330-335 (2003).</li></ol>

Die Autoren haben nichts zu offenbaren.

The HPLC purification of the A&#946; peptide is highly dependent upon the choice of both the stationary phase employed in the purification and the mobile phase chosen to elute the peptide. The low isoelectric point of the peptide and high propensity for aggregation render traditional chromatographic conditions for the separation of hydrophobic proteins (C4 or C8 stationary phase coupled with an acidic mobile eluent) challenging, with the A&#946; peptide eluting as a prolonged broad, non-resolved peak (Figure 2A).
To circumvent this issue, the PLRP-S stationary phase, chemically stable at high pH was found to be effective for the purification of the A&#946; peptide (Figure 2B and 2C). The use of a high pH mobile phase minimized the degree of aggregation, and when optimized, gave rise to a sharp, well-resolved peak. In the optimized protocol, the percentage of buffer B was rapidly switched from 20% to 27% at the 52-minute time point and as a result, gave rise to a well-defined A&#946; peak (Figure 2C). This optimized protocol relies on all of the hydrophobic impurities being eluted from the column during the initial 20 to 24.5% increase in buffer B concentration. If impurities are found which are of a similar hydrophobicity as the A&#946; peptide, then further optimization of the HPLC protocol may be warranted. The optimized method was highly reproducible amongst individual batches of synthesized A&#946; and was capable of purifying both 40 and 42 variants of the peptide without any changes to the optimized procedure (Figure 3). For both peptides, the purity as determined by analytical HPLC was found to be greater than 95%.
Given the reports of trace impurities altering the aggregation propensity of the A&#946; peptide, it is advisable that an orthogonal biophysical characterization be employed to confirm that the purified peptide is able to undergo oligomerization. Using previously reported protocols we chose to perform PICUP.14,15 PICUP analysis of the purified proteins demonstrates the characteristic monomer through hexamer population distribution for the A&#946;40 peptide and the monomer through heptamer distribution associated with the A&#946;42 peptide.10 These results confirm that purification of the A&#946; peptides using the PLRP-S stationary phase and a high pH mobile eluent results in A&#946; proteins that are capable of aggregation.
The purification procedure reported is designed to be able to purify up to 3 mg of the A&#946; peptide in a single chromatographic run. For this procedure, it is critical to set the flow rate of the HPLC instrument at 6 mL/min. If lower flow rates of the HPLC pump are used, the HPLC run time should be extended to accommodate this. Conversely, the use of higher flow rates may warrant a reduction in the HPLC run time. However, reduction of the run time can result in the co-elution of the A&#946; peak with other peaks and therefore lower the purity of the A&#946; peptide collected. Furthermore, the HPLC instrumentation and size of the chromatographic column used for purification can greatly affect the amount of material that can be purified in a single run. If no peak is produced at the expected eluent time of the A&#946; peptide, and a large peak is produced at the eluent time corresponding to the solvent front, then too much material was initially loaded onto the column. Reducing the amount of material injected onto the HPLC column for each run will circumvent this problem. Once the A&#946; peptide has been purified, it is highly recommended that the solution be lyophilized as quickly as possible. Prolonged storage of the peptide in solution can lead to oxidation of A&#946; and therefore formation of impurities. The purity of the A&#946; peptide should always be determined by analytical HPLC. Trace amounts of impurities can alter the aggregation propensity of the peptide dramatically. If the analytical HPLC trace shows the presence of impurities, then the sample should be re-subjected to the HPLC purification protocol and its purity re-determined by analytical HPLC.
It is hoped that this tailored procedure for the purification of the A&#946;42 and A&#946;40 peptides will be of benefit for the scientific community and allow users to obtain high purity A&#946; capable of oligomer formation. It would be expected that this procedure could be adapted to other amyloidogenic peptides that are difficult to isolate and purify.

Alzheimer-Krankheit ist eine neurodegenerative Erkrankung, die Auswirkungen über 35 Millionen Menschen weltweit. 1 stark bei der Entstehung und Entwicklung der Krankheit in Verbindung gebracht, ist die hoch Aggregation neigen, hydrophoben Peptid Amyloid beta (Aß). 2 Aß im Bereich von 36 bis 43 Aminosäuren in der Länge, aber es wird angenommen , daß die 42-Aminosäurevariante Amyloid beta 42 (Aß42), ist die toxische Form des Proteins. 3 Dies ist zum größten Teil auf die Fähigkeit von Aß42 zu leicht diffusionsfähig, oligomere Spezies bilden , die vermutlich besonders neurotoxischen Einheiten sein. 4 Um unser Verständnis des Aß - Peptid zu fördern, ist es wichtig , regelmäßig zu hochreinem Material zu erhalten. Das Vorhandensein von Spurenverunreinigungen hat sich gezeigt, dramatisch die Aggregationsneigung Eigenschaften des Peptids zu verändern. 5 Trüher, wurde durch die Verwendung einer Kombination von C 4 oder C 8 Siliciumdioxid basierenden stationären Phasen und einer sauren mobilen Phase die Hochleistungsflüssigkeitschromatographie (HPLC) -Trennung von hydrophoben Peptiden , wie beispielsweise Aß getan. 6 Jedoch können solche Bedingungen eine Herausforderung für die Reinigung des Peptids darstellen. Der niedrige isoelektrische Punkt des Aß - Peptid (pI ungefähr 5,5) , 7 bedeutet , daß unter sauren Bedingungen, Peptidaggregation erhöht wird und als Ergebnis breite, nicht aufgelöste HPLC - Peaks , die oft schwierig zu isolieren sind (2A) hergestellt. Ferner können solche breite Peaks enthalten oft Verunreinigungen, die die Aggregationsprofil des Peptids beeinflussen können, und erfordern häufig nachfolgenden Runden der Reinigung, die die Menge an Peptid dramatisch beeinflussen können hergestellt werden. Die Poly (Styrol / Divinylbenzol) stationäre Phase, PLRP-S stellt ein alternatives Mittel zur purifying hydrophobe Peptide. Die stationäre Phase wurde bei der Reinigung von einer Reihe von verschiedenen Proteinen und messenger-Ribonukleinsäuren (mRNA) eingesetzt werden. 8, 9 Die PLRP-S stationäre Phase erfordert keine zusätzliche Alkylliganden für Umkehrphasentrennung, und was noch wichtiger ist , bei hohen pH chemisch stabil, die Desaggregation des Peptids führt. 7 Hier berichten wir über eine maßgeschneiderte HPLC - Reinigung - Protokoll , das hochreine Beta - Amyloid-42 (Aß42) und Beta - Amyloid-40 (Aβ40) Peptide ergibt.

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		<tr height="38">
			<td height="38" style="height:38px;width:207px;">Agilent 1260 Infinity II quarternary pump</td>
			<td style="width:85px;">Agilent</td>
			<td style="width:179px;">G7111B</td>
			<td style="width:739px;">http://www.agilent.com/en-us/products/liquid-chromatography/lc-pumps-vacuum-degassers/1260-infinity-ii-quaternary-pump</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:207px;">Agilent 1260 Infinity II Dual variable wavelength detector</td>
			<td style="width:85px;">Agilent</td>
			<td style="width:179px;">G7114A</td>
			<td style="width:739px;">http://www.agilent.com/en-us/products/liquid-chromatography/lc-detectors/1260-infinity-ii-variable-wavelength-detector</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:207px;">Agilent 1260 Infinity II Manual Injector fitted with 10 mL stainless steel sample loop</td>
			<td style="width:85px;">Agilent</td>
			<td style="width:179px;">0101-1232</td>
			<td style="width:739px;">http://www.agilent.com/en-us/products/liquid-chromatography/lc-injection-systems/1260-infinity-ii-manual-injector</td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;width:207px;">Agilent 1260 Infinity II Manual Injector fitted with 20 &#181;L stainless steel sample loop</td>
			<td style="width:85px;">Agilent</td>
			<td style="width:179px;">G1328C</td>
			<td style="width:739px;">http://www.agilent.com/en-us/products/liquid-chromatography/lc-injection-systems/1260-infinity-ii-manual-injector</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:207px;">Ring Stand Mounting Bracket</td>
			<td style="width:85px;">Agilent</td>
			<td style="width:179px;">1400-3166</td>
			<td style="width:739px;"></td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:207px;">PEEK Tubing Blue (1/32&#34; outer diameter &#1093; 0.010&#34; internal diameter)</td>
			<td style="width:85px;">Thermo Scientific</td>
			<td style="width:179px;">03-050-399</td>
			<td style="width:739px;"></td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:207px;">Agilent PLRP-S 300&#197; 8&#181;m 25 &#1093; 300 mm column (Preparative)</td>
			<td style="width:85px;">Agilent</td>
			<td style="width:179px;">PL1212-6801</td>
			<td style="width:739px;">http://www.agilent.com/en-us/products/liquid-chromatography/lc-columns/biomolecule-separations/plrp-s-for-biomolecules#features</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:207px;">Agilent PLRP-S 300&#197; 8&#181;m 7.5 &#1093; 300 mm (Semi-Preparative)</td>
			<td style="width:85px;">Agilent</td>
			<td style="width:179px;">PL1112-6801</td>
			<td style="width:739px;">http://www.agilent.com/en-us/products/liquid-chromatography/lc-columns/biomolecule-separations/plrp-s-for-biomolecules#features</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:207px;">Agilent PLRP-S 300&#197; 5 &#181;m 4.6 x 250 mm (Analytical)</td>
			<td style="width:85px;">Agilent</td>
			<td style="width:179px;">PL1512-5501</td>
			<td style="width:739px;">http://www.agilent.com/en-us/products/liquid-chromatography/lc-columns/biomolecule-separations/plrp-s-for-biomolecules#features</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">A&#946;42 or A&#946;40 peptide</td>
			<td style="width:85px;"></td>
			<td style="width:179px;"></td>
			<td style="width:739px;">Synthesized in-house using a CEM liberty automated peptide synthesizer.</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:207px;">Ammonium Hydroxide (NH4OH, 28% solution)</td>
			<td style="width:85px;">Fisher Scientific</td>
			<td style="width:179px;">A669-500</td>
			<td style="width:739px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:207px;">Acetonitrile</td>
			<td style="width:85px;">Fisher Scientific</td>
			<td style="width:179px;">A998-4</td>
			<td style="width:739px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:207px;">HPLC grade water</td>
			<td style="width:85px;">Fisher Scientific</td>
			<td style="width:179px;">W5-4</td>
			<td style="width:739px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:207px;">Falcon 50 ml conical centrifuge tube</td>
			<td style="width:85px;">Fisher Scientific</td>
			<td style="width:179px;">14-954-49A</td>
			<td style="width:739px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:207px;">Supelco PEEK Fitting One-piece fingertight, pkg of 5 ea</td>
			<td style="width:85px;">Sigma-Aldrich</td>
			<td style="width:179px;">Z227250</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:207px;">Normject 5cc sterile syringe</td>
			<td style="width:85px;">Fisher Scientific</td>
			<td>1481729</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:207px;">16 Gauge SS Needle</td>
			<td style="width:85px;">Rheodyne</td>
			<td style="width:179px;">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.

Die Reinigung des Aß42 Peptid , das eine Kombination aus der PLRP-S stationäre Phase und ein hoher pH - Wert der mobilen Phase führt zur Bildung eines scharfen, aufgelöst Peak für das Aß - Peptid bei einer Retentionszeit zwischen 72 und 74 min (2C) verwendet wird . Bestätigung der Identität der Peaks wird durch direkte Injektion Massenspektrometrie des gesammelten Eluenten erfolgen. Der Eluent kann für bis zu 12 h bei -20 ° C in Lösung gelagert werden. Längere Lagerzeiten können bei der Oxidation des Proteins führen. Um das gereinigte Peptid zu isolieren, ist der Eluent in flüssigem Stickstoff eingefroren und lyophilisiert. Analytische HPLC des gereinigten Peptids zeigt eine Reinheit von&gt; 95% (Figur 2D). Das gleiche Verfahren kann verwendet werden , um die Aβ40 - Peptid (3A) ohne Modifikation des Verfahrens zu reinigen. Analytische HPLC zeigt eine Reinheit von Aβ40 zu mehr als 95% (3B). </p&gt; <img alt="Figur 3" src="/files/ftp_upload/55482/55482fig3.jpg" /> Abbildung 3: Repräsentative HPLC Spuren von Aβ40. (A) Optimierte präparative Reinigung der 25 x 300 mm 2 Polymer Spalte, Bedingungen sind in Tabelle 1 beschrieben , befindet sich in Unterabschnitt 1.3 des Protokolls Beschreibungstext. Die Peak-zu Aβ40 entspricht, mit einem Sternchen gekennzeichnet. (B) Analytische HPLC unter Verwendung der 4,6 x 250 mm 2 Polymer Spalte Bedingungen: Puffer A: H 2 O mit 20 mM NH 4 OH, Puffer B: 80% MeCN / 20% H 2 O mit 20 mM NH 4 OH, Gradient : 5 bis 50% Puffer B über 30 min. Sammlung des markierten Aβ40 peak in Teil A mit einer Reinheit von&gt; 95% ergibt , wie in Teil B. Massenspektrometrie gezeigt wurde verwendet , um die Identität des Aβ40 Peak zu bestimmen. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55482/55482fig3large.jpg" target="_blank">Bitte klicken sie</a>e eine größere Version dieser Figur zu sehen. Um zu bestätigen , daß das gereinigte Peptid oligomeren Mischungen bilden können, führten wir photoinduzierten Vernetzung des unmodifizierten Proteine (PICUP) , wie zuvor beschrieben , 14, 15 und konnten Oligomerbildung robust zu beobachten. 10

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

Wir berichten hier über eine HPLC-Reinigung Protokoll zugeschnitten, die hochreine Beta-Amyloid-42 (Aß42) und Beta-Amyloid-40 (Aβ40) Peptide, der fähig Oligomerbildung ergibt. Amyloid beta ist ein hoch Aggregation neigen, hydrophoben Peptid in Alzheimer-Krankheit in Verbindung gebracht. Die amyloidogenen Natur des Peptids macht seine Reinigung eine Herausforderung.

Ein Tailored HPLC Reinigungsprotokoll, dass die Renditen hochreine Amyloid Beta 42 und Amyloid Beta 40 Peptides, Fähig zur Oligomerbildung

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

Nanyang Technological University

Research

JoVE Journal

Biochemistry

11.8K Aufrufe.  University of California, Santa Cruz.  Wir berichten hier über eine HPLC-Reinigung Protokoll zugeschnitten, die hochreine Beta-Amyloid-42 (Aß42) und Beta-Amyloid-40 (Aβ40) Peptide, der fähig Oligomerbildung ergibt. Amyloid beta ist ein hoch Aggregation neigen, hydrophoben Peptid in Alzheimer-Krankheit in Verbindung gebracht. Die amyloidogenen Natur des Peptids macht seine Reinigung eine Herausforderung. 

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

Laboratory Scale Production and Purification of a Therapeutic Antibody

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the antibody genes followed by stable transfection into a suitable cell line. The stable clones are subjected to screening in order to select those clones with desired production and growth characteristics. This is a critical albeit time-consuming step in the process. This protocol considers vector selection and sourcing of antibody sequences for the expression of a therapeutic antibody. The methods describe preparation of vector DNA for stable transfection of a suspension variant of human embryonic kidney 293 (HEK-293) cell line, using polyethylenimine (PEI). The cells are transfected as adherent cells in serum-containing media to maximize transfection efficiency, and afterwards adapted to serum-free conditions. Large scale production, setup as batch overgrow cultures is used to yield antibody protein that is purified by affinity chromatography using an automated fast protein liquid chromatography (FPLC) instrument. The antibody yields produced by this method can provide sufficient protein to begin initial characterization of the antibody. This may include in vitro assay development or physicochemical characterization to aid in the time-consuming task of clonal screening for lead candidates. This method can be transferable to the development of an expression system for the production of biosimilar antibodies.

This protocol describes the production of a therapeutic antibody in a mammalian expression system. The methods described include preparation of vector DNA, stable transfection and serum-free adaptation of a human embryonic kidney 293 cell line, set up of large scale cultures and purification using affinity chromatography.

Labormaßstab Herstellung und Reinigung eines therapeutischen Antikörpers

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the ...

Forschung

Biochemie

Expression, Purification, and Antimicrobial Activity of S100A12

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some S100 proteins have the capacity to bind transition metals with high affinity and effectively sequester them away from invading microbial pathogens in a process that is termed "nutritional immunity". S100A12 (EN-RAGE) binds both zinc and copper and is highly abundant in innate immune cells such as macrophages and neutrophils. We report a refined method for the expression, enrichment and purification of S100A12 in its active, metal-binding configuration. Utilization of this protein in bacterial growth and viability analyses reveals that S100A12 has antimicrobial activity against the bacterial pathogen, Helicobacter pylori. The antimicrobial activity is predicated on the zinc-binding activity of S100A12, which chelates nutrient zinc, thereby starving H. pylori which requires zinc for growth and proliferation.

Here, we present a method to express and purify S100A12 (calgranulin C). We describe a protocol to measure its antimicrobial activity against the human pathogen H. pylori.

Expression, Reinigung und antimikrobielle Aktivität von S100A12

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some ...

Imaging Amyloid Tissues Stained with Luminescent Conjugated Oligothiophenes by Hyperspectral Confocal Microscopy and Fluorescence Lifetime Imaging

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find neurodegenerative diseases such as Alzheimer&#39;s and Parkinson&#39;s disease afflicting primarily the central nervous system, and systemic amyloidosis where serum amyloid A, transthyretin and IgG light chains deposit as amyloid in liver, carpal tunnel, spleen, kidney, heart, and other peripheral tissues. Amyloid has been known and studied for more than a century, often using amyloid specific dyes such as Congo red and Thioflavin T (ThT) or Thioflavin (ThS). In this paper, we present heptamer-formyl thiophene acetic acid (hFTAA) as an example of recently developed complements to these dyes called luminescent conjugated oligothiophenes (LCOs). hFTAA is easy to use and is compatible with co-staining in immunofluorescence or with other cellular markers. Extensive research has proven that hFTAA detects a wider range of disease associated protein aggregates than conventional amyloid dyes. In addition, hFTAA can also be applied for optical assignment of distinct aggregated morphotypes to allow studies of amyloid fibril polymorphism. While the imaging methodology applied is optional, we here demonstrate hyperspectral imaging (HIS), laser scanning confocal microscopy and fluorescence lifetime imaging (FLIM). These examples show some of the imaging techniques where LCOs can be used as tools to gain more detailed knowledge of the formation and structural properties of amyloids. An important limitation to the technique is, as for all conventional optical microscopy techniques, the requirement for microscopic size of aggregates to allow detection. Furthermore, the aggregate should comprise a repetitive &#946;-sheet structure to allow for hFTAA binding. Excessive fixation and/or epitope exposure that modify the aggregate structure or conformation can render poor hFTAA binding and hence pose limitations to accurate imaging.

Amyloid deposition is a hallmark of different diseases and afflicts many different organs. This paper describes the application of luminescent conjugated oligothiophene fluorescence staining in combination with fluorescence microscopy techniques. This staining method represents a powerful tool for detection and exploration of protein aggregates in both clinical and scientific setups.

Imaging-Amyloid Gewebe gebeizt mit lumineszierenden konjugierte Oligothiophenes von hyperspektralen konfokalen Mikroskopie und Fluoreszenz Lifetime Imaging

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find ...

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be greatly facilitated by the production of milligram amounts of pure, folded ligand binding domains. Attempts to heterologously express periplasmic ligand binding domains of bacterial chemoreceptors in Escherichia coli (E. coli) often result in their targeting into inclusion bodies. Here, a method is presented for protein recovery from inclusion bodies, its refolding and purification, using the periplasmic dCACHE ligand binding domain of Campylobacter jejuni (C. jejuni) chemoreceptor Tlp3 as an example. The approach involves expression of the protein of interest with a cleavable His6-tag, isolation and urea-mediated solubilisation of inclusion bodies, protein refolding by urea depletion, and purification by means of affinity chromatography, followed by tag removal and size-exclusion chromatography. The circular dichroism spectroscopy is used to confirm the folded state of the pure protein. It has been demonstrated that this protocol is generally useful for production of milligram amounts of dCACHE periplasmic ligand binding domains of other bacterial chemoreceptors in a soluble and crystallisable form.

A procedure is presented for the refolding of the dCACHE periplasmic ligand binding domain of Campylobacter jejuni chemoreceptor Tlp3 from inclusion bodies and the purification to yield milligram quantities of protein.

Methode für effiziente Umfaltung und Reinigung des Chemorezeptor-Liganden-bindende Domäne

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be ...

Use of Two Dimensional Semi-denaturing Detergent Agarose Gel Electrophoresis to Confirm Size Heterogeneity of Amyloid or Amyloid-like Fibers

Amyloid or amyloid-like fibers have been associated with many human diseases, and are now being discovered to be important for many signaling pathways. The ability to readily detect the formation of these fibers under various experimental conditions is essential for understanding their potential function. Many methods have been used to detect the fibers, but not without some drawbacks. For example, electron microscopy (EM), or staining with Congo Red or Thioflavin T often requires purification of the fibers. On the other hand, semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) allows detection of the SDS-resistant amyloid-like fibers in the cell extracts without purification. In addition, it allows the comparison of the size difference of the fibers. More importantly, it can be used to identify specific proteins within the fibers by Western blotting. It is less time consuming and more easily accessible to a wider number of labs. SDD-AGE results often show variable degree of heterogeneity. It raises the question whether part of the heterogeneity results from the dissociation of the protein complex during the electrophoresis in the presence of SDS. For this reason, we have employed a second dimension of SDD-AGE to determine if the size heterogeneity seen in SDD-AGE is truly a result of fiber heterogeneity in vivo and not a result of either degradation or dissociation of some of the proteins during electrophoresis. This method allows fast, qualitative confirmation that the amyloid or amyloid-like fibers are not partially dissociating during the SDD-AGE process.

Here we use two-dimensional semi-denaturing agarose gel electrophoresis to confirm the presence of amyloid-like fibers of heterogeneous size and exclude the possibility that the size heterogeneity is due to dissociation of the amyloid fibers during the gel running process.

Verwendung von zwei dimensionale Semi-denaturierenden Waschmittel Agarose Gelelektrophorese Größe Heterogenität von Amyloid oder Amyloid-wie Fasern zu bestätigen

Amyloid or amyloid-like fibers have been associated with many human diseases, and are now being discovered to be important for many signaling pathways. ...

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation of protein synthesis is the key to cellular stress-response, and dysregulation is central to many disease states, such as cancer. For instance, although cellular stress leads to the inhibition of global translation by attenuating cap-dependent initiation, certain stress-response proteins are selectively translated in a cap-independent manner. Discreet RNA regulatory elements, such as cellular internal ribosome entry sites (IRESes), allow for the translation of these specific mRNAs. Identification of such mRNAs, and the characterization of their regulatory mechanisms, have been a key area in molecular biology. Toeprinting is a method for the study of RNA structure and function as it pertains to translation initiation. The goal of toeprinting is to assess the ability of in vitro transcribed RNA to form stable complexes with ribosomes under a variety of conditions, in order to determine which sequences, structural elements, or accessory factors are involved in ribosome binding&#8212;a pre-cursor for efficient translation initiation. Alongside other techniques, such as western analysis and polysome profiling, toeprinting allows for a robust characterization of mechanisms for the regulation of translation initiation.

Toeprinting aims to measure the ability of in vitro transcribed RNA to form translation initiation complexes with ribosomes under a variety of conditions. This protocol describes a method for toeprinting mammalian RNA and can be used to study both cap-dependent and IRES-driven translation.

Toeprinting Analyse der Übersetzung Einleitung Komplexbildung auf Säugetier-mRNAs

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation ...

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-positiver β-Amyloid Oligomer Vorbereitung und Bewertung mit Dot-Blot-Analyse

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

Purification and Reconstitution of TRPV1 for Spectroscopic Analysis

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine and remain largely unknown. With recent advances in single-particle cryo-electron microscopy (cryo-EM) shedding light on the structural features of agonist binding sites and the activation mechanism of several ion channels, the stage is set for an in-depth dynamic analysis of their gating mechanisms using spectroscopic approaches. Spectroscopic techniques such as electron paramagnetic resonance (EPR) and double electron-electron resonance (DEER) have been mainly restricted to the study of prokaryotic ion channels that can be purified in large quantities. The requirement for large amounts of functional and stable membrane proteins has hampered the study of mammalian ion channels using these approaches. EPR and DEER offer many advantages, including determination of the structure and dynamic changes of mobile protein regions, albeit at low resolution, that might be difficult to obtain by X-ray crystallography or cryo-EM, and monitoring reversible gating transition (i.e., closed, open, sensitized, and desensitized). Here, we provide protocols for obtaining milligrams of functional detergent-solubilized transient receptor potential cation channel subfamily V member 1 (TRPV1) that can be labeled for EPR and DEER spectroscopy.

This article describes specific methods to obtain biochemical quantities of detergent-solubilized TRPV1 for spectroscopic analysis. The combined protocols provide biochemical and biophysical tools that can be adapted to facilitate structural and functional studies for mammalian ion channels in a membrane-controlled environment.

Reinigung und Wiederherstellung der TRPV1 für spektroskopische Analyse

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine ...

Detection and Isolation of Apoptotic Bodies to High Purity

Apoptotic bodies (ApoBDs), microvesicles and exosomes are the key members of the extracellular vesicle family, with ApoBDs being one of the largest type. It has been proposed that ApoBDs can aid cell clearance as well as intercellular communication through trafficking biomolecules. Conventional approaches used for the identification and isolation of ApoBDs are often limited by the lack of accurate quantification and low sample purity. Here, we describe a workflow to confirm the induction of apoptosis, validate ApoBD formation, and isolate ApoBDs to high purity. We will also outline and compare fluorescence-activated cell sorting (FACS) and differential centrifugation based approaches to isolate ApoBDs. Furthermore, the purity of isolated ApoBDs will be confirmed using a previously establish flow cytometry-based staining and analytical method. Taken together, using the described approach, THP-1 monocyte apoptosis and apoptotic cell disassembly was induced and validated, and ApoBD generated from THP-1 monocytes were isolated to a purity of 97-99%.

A workflow using flow cytometry or differential centrifugation is developed to detect, quantify and isolate apoptotic bodies from an apoptotic sample to high purity.

Nachweis und Isolierung von apoptotischen stellen hohe Reinheit

Apoptotic bodies (ApoBDs), microvesicles and exosomes are the key members of the extracellular vesicle family, with ApoBDs being one of the largest type. ...

Analysis of &#946;-Amyloid-induced Abnormalities on Fibrin Clot Structure by Spectroscopy and Scanning Electron Microscopy

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A&#946;) protein on clot formation and structure by spectrometry and scanning electron microscopy (SEM). A&#946;, which forms neurotoxic amyloid aggregates in Alzheimer&#39;s disease (AD), has been shown to interact with fibrinogen. This A&#946;-fibrinogen interaction makes the fibrin clot structurally abnormal and resistant to fibrinolysis. A&#946;-induced abnormalities in fibrin clotting may also contribute to cerebrovascular aspects of the AD pathology such as microinfarcts, inflammation, as well as, cerebral amyloid angiopathy (CAA). Given the potentially critical role of neurovascular deficits in AD pathology, developing compounds which can inhibit or lessen the A&#946;-fibrinogen interaction has promising therapeutic value. In vitro methods by which fibrin clot formation can be easily and systematically assessed are potentially useful tools for developing therapeutic compounds. Presented here is an optimized protocol for in vitro generation of the fibrin clot, as well as analysis of the effect of A&#946; and A&#946;-fibrinogen interaction inhibitors. The clot turbidity assay is rapid, highly reproducible and can be used to test multiple conditions simultaneously, allowing for the screening of large numbers of A&#946;-fibrinogen inhibitors. Hit compounds from this screening can be further evaluated for their ability to ameliorate A&#946;-induced structural abnormalities of the fibrin clot architecture using SEM. The effectiveness of these optimized protocols is demonstrated here using TDI-2760, a recently identified A&#946;-fibrinogen interaction inhibitor.

Presented here are two methods that can be used individually or in combination to analyze the effect of beta-amyloid on fibrin clot structure. Included is a protocol for creating an in vitro fibrin clot, followed by clot turbidity and scanning electron microscopy methods.

Analyse der β-Amyloid-induzierte Anomalien auf Fibrin Gerinnsel Struktur von Spektroskopie und Rasterelektronenmikroskopie

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A&#946;) protein on clot formation and ...