Name: a tailored hplc purification protocol that yields high-purity amyloid beta 42 and amyloid beta 40 peptides, capable of oligomer formation
Uploaded: 2017-03-27T14:00:00
Description: 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

Författarna vill tacka Agilent för deras tekniskt stöd. Kate Markham och Rafael Palomino kredit för sin första hjälp i syntesen och reningen av Ap-peptiden och Dr Hsiau-Wei Lee tackade för hans hjälp att förbereda Figur 1 av manuskriptet.

1. Preparativ HPLC Rening av Aβ40 eller Aβ42 peptid <ol><li> Förbered följande buffertar för HPLC-rening. <ol><li> Förbereda buffert A (20 mM NH4OH) genom att tillsätta 1,3 ml NH4OH (28% lösning) till 1000 ml ultrarent vatten. </li><li> Förbereda buffert B (80% acetonitril med 20 mM NH4OH) genom att tillsätta 1,3 ml NH4OH (28% lösning) till en lösning av 800 ml av HPLC-kvalitet acetonitril och 200 ml ultrarent vatten. </li><li> Förbereda provupp...

<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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HPLC-rening av Ap-peptiden är mycket beroende på valet av både den stationära fasen som användes vid reningen och den mobila fasen väljs för att eluera peptiden. Den låga isoelektriska punkten för peptiden och hög benägenhet för aggregering render traditionella kromatografiska förhållanden för separation av hydrofoba proteiner (C4 eller C8 stationär fas i kombination med en sur mobil elueringsmedel) utmanande, med Ap-peptiden eluerades som en förlängd bred, icke-löst topp (Figur 2A). ...

Alzheimers sjukdom är en neurodegenerativ sjukdom som effekter över 35 miljoner människor världen över. 1 Inblandad kraftigt i början och utveckling av sjukdomen, är den mycket sammanläggning liggande, hydrofoba peptid Amyloid beta (AP). 2 Ap varierar från 36 till 43 aminosyror i längd, dock är det tänkt att den 42-aminosyra-variant, amyloid beta 42 (Aβ42), är den giftigaste formen av proteinet. 3 Detta beror till största delen på förmågan hos Aβ42 att lätt bilda diffunderbara, oligomera arter som anses vara särskilt neurotoxiska enheter. 4 För att öka vår förståelse av Ap-peptiden, är det viktigt att rutinmässigt erhålla hög renhet material. Närvaron av spår av föroreningar har visat sig dramatiskt förändra aggregering propensity egenskaperna hos peptiden. 5 Traditionally har den högupplösande vätskekromatografi (HPLC) separation av hydrofoba peptider, såsom Ap gjorts genom användning av en kombination av C 4 eller C 8 kiseldioxidbaserade stationära faser och en sur mobil fas. 6 kan dock sådana förhållanden utgör en utmaning för rening av peptiden. Den låga isoelektriska punkten för Ap-peptiden (pl ungefär 5,5) 7 innebär att under sura betingelser, är peptid aggregation ökas och som ett resultat breda, icke-upplösta HPLC-toppar som ofta är svåra att isolera produceras (Figur 2A). Dessutom sådana breda toppar innehåller ofta föroreningar som kan påverka aggregering profil av peptiden, och vanligtvis kräver efterföljande omgångar av rening som dramatiskt kan påverka mängden peptid som produceras. Poly (styren / divinylbensen) stationär fas, PLRP-S, representerar ett alternativt sätt att purifying hydrofoba peptider. Den stationära fasen har varit anställd vid reningen av ett antal olika proteiner och budbärare ribonukleinsyror (mRNA). 8, kräver 9 Den PLRP-S stationär fas ingen ytterligare alkyl ligand för omvänd fasseparation, och ännu viktigare är kemiskt stabil vid högt pH vilket leder till aggregatupplösning av peptiden. 7 Häri rapporterar vi en skräddarsydd HPLC reningsprotokoll som ger hög renhet amyloid beta 42 (Aβ42) och amyloid beta 40 (Aβ40) peptider.

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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.

Reningen av den Aβ42 peptiden med användning av en kombination av den PLRP-S stationär fas och ett högt pH mobil fas resulterar i bildandet av en skarp, löst toppen för den Ap-peptiden vid en retentionstid mellan 72 och 74 min (figur 2C). Bekräftelse av identiteten av toppen sker genom direktinsprutning masspektrometri av den uppsamlade elueringsmedlet. Elueringsmedlet kan lagras vid -20 ° C i lösning i upp till 12 timmar. Längre perioder av lagring kan leda ti...

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

Häri rapporterar vi en skräddarsydd HPLC reningsprotokoll som ger hög renhet amyloid beta 42 (Aβ42) och amyloid beta 40 (Aβ40) peptider som kan oligomerbildning. Amyloid beta är en i hög grad aggregation benägen, hydrofob peptid inblandad i Alzheimers sjukdom. Den amyloidogeniska karaktären av peptiden gör dess rening en utmaning.

En skräddarsydd HPLC reningsprotokoll som ger hög renhet Amyloid Beta 42 och Amyloid Beta 40 Peptider som kan oligomerbildning

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

The overall goal of this procedure is to use HPLC purification to produce high purity amyloid beta 42, and amyloid beta 40 peptides that are capable of forming oligomers.The biochemical properties of the amyloid beta peptide can often make its purification difficult.Therefore, the obtainment of high purity peptide that is capable of oligomer formation can be challenging.This procedure helps to alleviate that challenge.For this HPLC protocol, have prepared stocks of buffers A and B ready as well as the buffer for sample dissolution.To set up the HPLC, first fit the bottles of buffer A and B to the inlets of the pump.Use polymer tubing and one piece fittings and don't mix up the valves.Connect the polymer tubing from the pump outlet to the inlet of the preparative column.Make the one piece fitting finger tight, and make certain the column is in the correct orientation.Now secure another polymer tube from the outlet of the column to the dual wavelength detector.Use finger tight one piece fittings, then, in the HPLC setup software, set the wavelength detector to measure peaks at 214 and 280 nanometers.Next, connect the output valve of the wave length detector to the outlet valve of the HPLC detector using polymer tubing and a one piece fitting made finger tight.This will serve as the sample collection tubing.Now program the HPLC software to run the purification method.From the Set up Instrument Method option, enter the purification method by changing the solvent timetable parameter.Lastly, turn on the HPLC pump from the HPLC software.Now the pump will start supplying the starting ratio of buffer A and buffer B through the preparative column in the HPLC instrument.It is vital to now let the system equilibrate for 30 minutes before proceeding with samples.To begin, dissolve three milligrams of crude A

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