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 ønsker å takke Agilent for deres teknisk assistanse. Kate Markham og Rafael Palomino er kreditert for sin første hjelp i syntese og rensing av Ap peptid og Dr Hsiau-Wei Lee takkes for hans hjelp i forberedelsene Figur 1 av manuskriptet.

1. Preparativ HPLC Rensing av Aβ40 eller Aβ42 Peptide <ol><li> Forbered følgende buffere for HPLC rensing. <ol><li> Forbered buffer A (20 mM NH4OH) ved å tilsette 1,3 ml NH4OH (28% løsning) til 1000 ml med ultrarent vann. </li><li> Forbered buffer B (80% acetonitril med 20 mM NH4OH) ved å tilsette 1,3 ml NH4OH (28% løsning) til en løsning av 800 ml av HPLC-kvalitet acetonitril og 200 ml ultrarent vann. </li><li> Fremstille prøveoppløsningsbuffer (0...

<ol>
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HPLC-rensing av Ap-peptidet er i stor grad avhengig av valget av både den stasjonære fasen som anvendes ved rensingen og den mobile fase valgt for å eluere peptidet. Den lave isoelektriske punkt av peptidet og høy tilbøyelighet til aggregering gjengi tradisjonelle kromatografiske betingelser for separering av hydrofobe proteiner (C4 eller C8 stasjonær fase kombinert med en sur mobil elueringsmiddel) utfordrende, med Ap-peptid ved å eluere som en forlenget bred, ikke løst peak (figur 2A). <p ...

Alzheimers sykdom er en nevrodegenerativ lidelse som effekter over 35 millioner mennesker verden over. 1 Impliserte sterkt i begynnelsen og utviklingen av sykdommen, er det svært utsatt aggregering, hydrofobt peptid Amyloid beta (Ap). 2. Ap varierer fra 36 til 43 aminosyrer i lengde, er det imidlertid antatt at en 42-aminosyre-variant, amyloid beta 42 (Aβ42), er det mest giftige formen av proteinet. 3 Dette skyldes i hovedsak til evnen til Aβ42 til lett danne diffusible, oligomeric arter som antas å være spesielt nevrotoksiske enheter. 4 For å fremme forståelsen av Ap peptid, er det viktig å rutinemessig oppnå høy renhet materiale. Tilstedeværelsen av sporforurensninger har vist seg å dramatisk endre aggregering tilbøyelighet egenskapene til peptidet. 5 Traditionally har høy ytelse væskekromatografi (HPLC) separering av hydrofobe peptider slik som Ap blitt gjort ved bruk av en kombinasjon av C-4 eller C-8 silika-baserte stasjonære faser og en sur mobil fase. 6 kan imidlertid slike forhold være en utfordring for rensing av peptidet. Den lave isoelektriske punktet til Ap peptidet (pI ca. 5,5) 7 betyr at under sure betingelser, er peptidaggregering øket, og som et resultat av brede, ikke-oppløste HPLC-topper som ofte er vanskelig å isolere blir produsert (figur 2A). Videre er slike brede topper inneholder ofte urenheter som kan påvirke aggregering profilen av peptidet, og som vanligvis krever påfølgende runder av rensing som kan dramatisk påvirke mengden av peptidet produsert. Poly (styren / divinylbenzen) stasjonær fase, PLRP-S, representerer en alternativ metode for vannrensingying hydrofobe peptider. Den stasjonære fase er blitt anvendt i rensingen av en rekke forskjellige proteiner og messenger ribonukleinsyrer (mRNA). 8, krever 9 PLRP-S stasjonær fase uten ekstra alkyl ligand for reversfaseseparasjon, og enda viktigere er kjemisk stabilt ved høy pH, hvilket fører til deaggregation av peptidet. 7 Heri, rapporterer vi en skreddersydd HPLC rensing protokoll som gir høy renhet 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.

Rensing av Aβ42 peptidet ved anvendelse av en kombinasjon av den PLRP-S-stasjonær fase og en høy pH-mobil fase resulterer i dannelse av en skarp, løst topp for Ap-peptid ved en oppholdstid mellom 72 og 74 min (figur 2C). Bekreftelse av identiteten av toppen skjer ved direkte innsprøyting av massespektrometri av den oppsamlede elueringsmiddel. Elueringsmidlet kan oppbevares ved -20 ° C i oppløsning i opptil 12 timer. Lengre tids lagring kan føre til oksidasjon av ...

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 vi rapportere en skreddersydd HPLC rensing protokoll som gir høy renhet amyloid beta 42 (Aβ42) og amyloid beta 40 (Aβ40) peptider, i stand til oligomerdannelsen. Amyloid beta er en svært aggregering utsatt, hydrofobe peptid innblandet i Alzheimers sykdom. Den amyloidogene natur av peptidet som gjør dets rensing en utfordring.

En Tailored HPLC rensing protokoll som gir høy renhet amyloid beta 42 og amyloid beta 40 peptider, i stand til oligomerdannelsen

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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