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

Yazarlar teknik yardım için Agilent teşekkür etmek istiyorum. Kate Markham ve Rafael Palomino Dr Hsiau-Wei Lee yazının Şekil 1 hazırlanmasında yaptığı yardım için teşekkür edilmektedir Aβ peptid ve onların ilk sentezinde yardım ve arıtma için yatırılır.

Aβ40 ya Aβ42 Peptid 1. Hazırlayıcı HPLC saflaştırma <ol><li> HPLC saflaştırması için, aşağıdaki tamponlar hazırlamak. <ol><li> Ultra saf su 1000 ml NH4OH (% 28 çözelti) 1.3 mL ilave edilerek tampon A (20 mM NH4OH) hazırlayın. </li><li> HPLC-grade asetonitril 800 mL ve ultra saf su 200 mL çözeltisine NH4OH (% 28 çözelti) 1.3 mL ilave edilerek tampon B (20 mM NH4 OH ile% 80 asetonitril) hazırlayın. </li><li> Ultra saf su ve 100 mL <sub...

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Aβ peptidi HPLC saflaştırma arıtılmadan kullanılan durağan faz peptid elüt edilmesi için seçilen mobil faz iki seçimi oldukça bağlıdır. toplama peptidin yüksek eğilimi düşük izoelektrik noktası Aβ peptidi uzun bir geniş olmayan çözüldü olarak elüsyon ile hidrofobik proteinlerin ayrılması (C4 veya asidik bir mobil elüsyon ile birleştirilmiş C8 faz) zor geleneksel kromatografik koşullan sağlamaya pik (Şekil 2A). Yüksek pH değerinde kimyasa...

Alzheimer hastalığı bir nörodejeneratif bir hastalıktır etkileri 35 milyondan fazla kişi dünya çapında. Hastalığın başlangıcı ve gelişiminde güçlü Belirtilmiş 1, yüksek toplama eğilimli, hidrofobik peptid Amiloid beta (Aβ) &#39;dir. 2 Aβ Ancak, beta amiloid 42 (Aβ42), proteinin en toksik bir şekilde 42-amino asit türevi olduğu düşünülmektedir, uzunluğu 36 ila 43 amino asit arasında değişir. 3 Bu hali hazırda, özellikle nörotoksik maddeler olduğu düşünülmektedir yayılabilir oligomerik türler oluşturma Aβ42 yeteneğini büyük bir kısmında kaynaklanmaktadır. 4 Aβ peptid anlayışımızı ilerletmek için, rutin yüksek saflıkta malzeme elde etmek esastır. saf olmayan artıklarının varlığı büyük ölçüde peptidin birikme eğilimi özelliklerini değiştirmek için gösterilmiştir. 5 Traditionally, örneğin Aβ hidrofobik peptitler, yüksek performanslı sıvı kromatografisi (HPLC) ayırma C 4 ya da Cı 8 silis bazlı sabit fazlarda ve bir asidik mobil faz bir kombinasyonunun kullanımı ile yapılmıştır. 6 Ancak, bu tür koşullar peptid saflaştırılması için bir meydan okuma sunabilir. Aβ peptidi düşük izoelektrik noktası (pl yaklaşık 5.5) 7, asidik koşullar altında, peptid agregasyon artar ve bunun bir sonucu olarak genellikle izole etmek zordur, geniş olmayan çözüldü HPLC tepe (Şekil 2A) üretilmeleri anlamına gelir. Ayrıca, bu tür geniş zirveler genellikle peptid toplama profilini etkileyebilecek ve yaygın dramatik üretilen peptid miktarını etkileyebilir arınma sonraki mermi gerektirebilir safsızlıklar içermezler. poli (stiren / divinilbenzen) durağan faz, PLRP-S, saflaştırmadan bir alternatif araçları temsil ederYing hidrofobik peptitler. hareketsiz faz farklı protein ve haberci ribonükleik asitlerin (mRNA) &#39;in bir dizi saflaştırılmasında kullanılmıştır. 8, 9 PLRP-S sabit faz ters faz ayrılması için ek bir alkil ligandı gerektirir ve daha da önemlisi, peptidin agregadan arındırma çaresinin yol açan yüksek pH değerinde kimyasal olarak kararlıdır. 7 Burada, yüksek saflıkta amiloid beta 42 (Aβ42) ve amiloid beta 40 (Aβ40) peptidleri veren bir özel HPLC arıtma protokolü sunduk.

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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.

PLRP-S durağan faz bir kombinasyonunu 72 ve 74 dakika boyunca (Şekil 2C) arasında bir alıkonma zamanında Aβ peptidi için keskin çözüldü tepe oluşumunda yüksek bir pH mobil faz sonuçları kullanılarak Aβ42 peptidinin arıtılması. tepe kimliğinin teyidi toplanan elüsyon direkt enjeksiyonu kütle spektrometrisi yoluyla yapılır. yıkama sıvısı kadar 12 saat boyunca çözelti içinde -20 ° C&#39;de muhafaza edilebilir. depolamanın daha uzun süreli ...

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

Bu yazıda oligomer oluşturabilen yüksek saflıkta amiloid beta 42 (Aβ42) ve amiloid beta 40 (Aβ40) peptidler, veren bir özel HPLC arıtma protokolü sunduk. Amiloid P Alzheimer hastalığında rol yüksek çekiş eğilimli hidrofobik peptittir. peptid amiloidojenik doğası onun saflaştırma bir meydan okuma yapar.

Bir Ismarlama HPLC Arıtma Protokolü That Oligomer Formasyonu Yetenekli Verim Yüksek saflıkta Amiloid Beta 42 ve Amiloid Beta 40 peptidler,

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

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

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