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

Forfatterne vil gerne takke Agilent for deres tekniske assistance. Kate Markham og Rafael Palomino er krediteret for deres første hjælp i syntese og rensning af Ap-peptid og Dr. Hsiau-Wei Lee takkede for hans hjælp i forberedelsen Figur 1 af manuskriptet.

1. Præparativ HPLC Oprensning af Aβ40 eller Aβ42 Peptid <ol><li> Fremstille følgende puffere til HPLC oprensning. <ol><li> Forbered puffer A (20 mM NH4OH) ved tilsætning 1,3 ml NH4OH (28% opløsning) til 1.000 ml ultrarent vand. </li><li> Forbered puffer B (80% acetonitril med 20 mM NH4OH) ved tilsætning 1,3 ml NH4OH (28% opløsning) til en opløsning af 800 ml HPLC-kvalitet acetonitril og 200 ml ultrarent vand. </li><li> Forbered prøve opløsning (0,...

<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. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+reversed-phase+high-performance+liquid+chromatography+on+polystyrene-divinylbenzene+columns+for+the+rapid+separation+and+purification+of+acid-soluble+nuclear+proteins.">Use of reversed-phase high-performance liquid chromatography on polystyrene-divinylbenzene columns for the rapid separation and purification of acid-soluble nuclear proteins.</a> J Chromatogr A. 763 (1-2), 65-70 (1997).</li><li>Thess, A., 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=Sequence-engineered+mRNA+Without+Chemical+Nucleoside+Modifications+Enables+an+Effective+Protein+Therapy+in+Large+Animals.">Sequence-engineered mRNA Without Chemical Nucleoside Modifications Enables an Effective Protein Therapy in Large Animals.</a> Mol Ther. 23 (9), 1456-1464 (2015).</li><li>Warner, C. 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HPLC oprensning af Ap-peptidet er meget afhængig af valget af både den stationære fase i oprensningen og den mobile fase valgt at eluere peptidet. Den lave isoelektriske punkt af peptidet og høj tilbøjelighed til aggregation gøre traditionelle kromatografiske betingelser for separation af hydrofobe proteiner (C4 eller C8 stationær fase kombineret med en sur mobil eluent) udfordrende, med Ap-peptidet eluering som en forlænget bred, ikke-løst top (figur 2A). For at om...

Alzheimers sygdom er en neurodegenerativ lidelse, effekter over 35 millioner mennesker verden over. 1 Impliceret kraftigt i indtræden og udvikling af sygdommen, er den meget sammenlægning tilbøjelige, hydrofobt peptid amyloid beta (Ap). 2 Ap spænder fra 36 til 43 aminosyrer i længde, imidlertid antages det, at den 42-aminosyrevariant, amyloid beta 42 (AP42), er den mest toksiske form af proteinet. 3 Dette skyldes i de fleste dele til evnen af Ap42 til let at danne diffunderbare, oligomere arter, som menes at være særligt neurotoksiske enheder. 4 For at fremme vores forståelse af Ap-peptidet, er det vigtigt at rutinemæssigt opnå høj renhed materiale. Tilstedeværelsen af ​​sporurenheder er blevet vist at dramatisk ændre aggregering tilbøjelighedsdata egenskaber af peptidet. 5 Traditionally har højtydende væskekromatografi (HPLC) separation af hydrofobe peptider, såsom Ap blevet gjort ved anvendelse af en kombination af C 4 eller C 8 silicabaserede stationære faser og en sur mobil fase. 6 Imidlertid kan sådanne betingelser udgør en udfordring for oprensningen af peptidet. Den lave isoelektriske punkt Ap-peptidet (pi ca. 5,5) 7 betyder, at under sure betingelser, er peptidaggregering øges, og som et resultat brede, ikke-opløste HPLC toppe, som ofte er vanskeligt at isolere produceres (figur 2A). Endvidere har sådanne brede toppe indeholder ofte urenheder, som kan påvirke sammenlægning profil af peptidet, og almindeligvis kræver efterfølgende runder af oprensning som dramatisk kan påvirke mængden af ​​peptid produceres. Poly (styren / divinylbenzen) stationær fase, PLRP-S, repræsenterer et alternativt middel til Purifying hydrofobe peptider. Den stationære fase er blevet anvendt i oprensningen af ​​en række forskellige proteiner og messenger ribonukleinsyrer (mRNA). 8, 9 PLRP-S stationære fase kræver ingen yderligere alkyl ligand for omvendt fase separation, og endnu vigtigere er kemisk stabilt ved høj pH, som fører til deaggregering af peptidet. 7 Heri rapporterer vi en skræddersyet HPLC-oprensning protokol, der giver høj renhed amyloid beta 42 (Aβ42) og 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.

Oprensningen af Aβ42-peptidet under anvendelse af en kombination af PLRP-S stationær fase og en høj pH-resultater mobil fase i dannelsen af en skarp, løst top for Ap-peptidet ved en retentionstid mellem 72 og 74 min (figur 2C). Bekræftelse af identiteten af ​​toppen sker ved direkte injektion massespektrometri af den indsamlede eluent. Eluenten kan opbevares ved -20 ° C i opløsning i op til 12 timer. Længere tids opbevaring kan resultere i oxidation af protei...

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

Heri rapporterer vi en skræddersyet HPLC-oprensning protokol, der giver høj renhed amyloid beta 42 (Aβ42) og amyloid beta 40 (Aβ40) peptider, der kan oligomerdannelse. Amyloid beta er et stærkt sammenlægning tilbøjelige, hydrofobt peptid impliceret i Alzheimers sygdom. Det amyloidogene natur af peptidet gør dets oprensning en udfordring.

En skræddersyet HPLC Oprensning protokol, der giver høj renhed Amyloid Beta 42 og Amyloid Beta 40 Peptider, der kan 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 ...

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