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. Preparative HPLC Purification of the A&#946;40 or A&#946;42 Peptide
<ol>
	<li>Prepare the following buffers for the HPLC purification.
	<ol>
		<li>Prepare buffer A (20 mM NH4OH) by adding 1.3 mL of NH4OH (28% solution) to 1,000 mL of ultrapure water.</li>
		<li>Prepare buffer B (80% acetonitrile with 20 mM NH4OH) by adding 1.3 mL of NH4OH (28% solution) to a solution of 800 mL of HPLC-grade acetonitrile and 200 mL of ultrapure water.</li>
		<li>Prepare sample dissolution buffer (0.1% NH4OH) by adding 100 &#181;L of NH4OH (28% solution) to 100 mL of ultrapure water.</li>
	</ol>
	</li>
	<li>Setup the HPLC instrument as shown in Figure 1.
	<ol>
		<li>Fit the solvent bottles that contain buffer A and buffer B to the inlets of the HPLC pump using polymer tubing. Attach the polymer tubing to the HPLC pump with a one-piece fitting. Ensure that the polymer tubing of each buffer is fitted to the correct inlet valve of the instrument. Fit the HPLC pump with a degasser.</li>
		<li>Couple the HPLC pump with the inlet of the 300 &#197; 8 &#181;m 25 mm &#1093; 300 mm preparative column (Figure 1B, far left column, see Materials List) using polymer tubing.
		<ol>
			<li>Attach the polymer tubing to the preparative column with a one-piece finger tight fitting. Ensure that the polymer column is orientated in the correct manner. 
			NOTE: The stationary phase of the preparative column is comprised of poly(styrene-divinylbenzene) particles. The correct orientation of the polymer column is marked on the outer casing with a single directional arrow.</li>
		</ol>
		</li>
		<li>Connect the outlet of the column to the dual wavelength detector using polymer tubing and set the wavelength detector to 214 nm and 280 nm.
		<ol>
			<li>Alter the wavelength by changing the detection wavelength parameters in the setup instrument method option of the built-in HPLC software.</li>
		</ol>
		</li>
		<li>Attach the polymer tubing to the inlet of the wavelength detector with a one-piece finger tight fitting. Attach polymer tubing to the output valve of the wavelength detector. Attach the polymer tubing to the outlet valve of the HPLC detector with a one-piece finger tight fitting. This will be the sample collection tubing. 
		NOTE: The lack of a strong chromophore on the A&#946; peptide dictates that 214 nm be used as the primary ultraviolet (UV) wavelength for peak collection.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/55482/55482fig1.jpg" /> 
Figure 1: Experimental setup of the HPLC instrument used for purification of the amyloid beta peptides. (A) The quaternary HPLC pump fitted with a degasser and variable wavelength detector set to 214 nm and 280 nm; (B) HPLC columns used for purification of the amyloid beta peptides, from left to right, 25 x 300 mm2 preparative column, 7.5 x 300 mm2 semi preparative column and 4.6 x 250 mm2 analytical column; (C) Manual injector with 20 &#181;L stainless steel injection loop used for analytical HPLC; (D) Manual injector with 10 mL stainless steel injection loop used for preparative and semi preparative purification. <a href="http://cloudfront.jove.com/files/ftp_upload/55482/55482fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="3">
	<li>Program the HPLC software to run the purification method as shown in Table 1. Enter the purification method by changing the solvent timetable parameter (in the setup instrument method option built into the HPLC software). Turn on the HPLC pump by clicking the &#34;on&#34; button on the HPLC software. 
	NOTE: The pump will begin supplying starting ratio of buffer A and buffer B through the preparative column and the HPLC instrument.
	<ol>
		<li>Leave the system for 30 min to fully equilibrate.</li>
	</ol>
	</li>
</ol>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Time / min</td>
			<td>% of Buffer Aa</td>
			<td>% of Buffer Bb</td>
			<td>Flow Rated / 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>95c</td>
			<td>6</td>
		</tr>
	</tbody>
</table>
Table 1: Timetable for the purification of the A&#946;42 and A&#946;40 peptides using the 25 &#215; 300 mm polymer column. aBuffer A - H2O with 20 mM NH4OH; b80% MeCN / 20% H2O with 20 mM NH4OH; cRan for 15 min to wash the column prior to the next injection of sample; dIn order to run a flow rate of 6 mL/min on the HPLC instrumentation, the pressure limit needs to be reduced to 200 bar.
<ol start="4">
	<li>Purification of the A&#946; peptide sample 
	Note: The crude peptide was obtained through automated solid-phase peptide synthesis.10
	<ol>
		<li>Dissolve 3 mg of crude A&#946; peptide in 4 mL of the sample dissolution buffer. Sonicate the sample for 30-60 s at room temperature and at a frequency of 40 kHz to aid dissolution.</li>
		<li>Inject the entire sample onto the HPLC column using a 5 mL plastic syringe fitted with a 16-gauge stainless steel needle. Run the purification method as outlined in sub-step 1.3. 
		NOTE: The system enables sample injection to be done through the use of a manual injector fitted with a 10 mL stainless steel injection loop (Figure 1D). The desired A&#946; peptide will elute between 72 and 74 min as a sharp resolved peak (Figure 2C).</li>
		<li>Collect the sample into a 50 mL conical centrifuge tube. Confirm the identity of the A&#946; peak through direct injection mass spectrometry of the collected eluent.11 Store the eluent for up to 12 h at -20 &#176;C. 
		NOTE: Storage of the solution for periods longer than 12 h is not advised due to the potential for oxidation of the peptide.</li>
		<li>Isolate the purified peptide by flash freezing the collected aliquot/aliquots of the A&#946; peptide in liquid nitrogen and lyophilize. Perform lyophilization by freeze-drying the sample at a temperature of -60 &#176;C and a pressure of 20 mTorr for a period of 24 h.</li>
		<li>Run the analytical HPLC protocol as outlined below to determine the purity of the A&#946; peptide. Store peptides in their lyophilized form at -20 &#176;C for a period of up to 6 months.</li>
	</ol>
	</li>
</ol>
2. Analytical HPLC Analysis of the Purified A&#946; Protein
<ol>
	<li>Prepare the HPLC buffers as outlined in sub-section 1.1. of the above protocol.</li>
	<li>Setup the analytical HPLC as per step 1.2.1 and as depicted in Figure 1 with the 4.6 &#215; 250 mm analytical column (Figure 1B, far right column) and the manual injector with 20 &#181;L stainless steel injection loop (Figure 1C) fitted to the instrument.</li>
	<li>Program the HPLC software to run the analytical method as shown in Table 2 following instructions similar to those in step 1.3.</li>
</ol>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Time / min</td>
			<td>% of Buffer Aa</td>
			<td>% of Buffer Bb</td>
			<td>Flow Rate / 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>
Table 2: Timetable for the HPLC purity analysis of the A&#946; peptide. aBuffer A - H2O with 20 mM NH4OH; b80% MeCN / 20% H2O with 20 mM NH4OH.
<ol start="4">
	<li>Purity analysis of the A&#946; peptide
	<ol>
		<li>Prepare a 1 mg/mL solution of the purified peptide through dissolution of the peptide in the sample buffer solution. 
		NOTE: The buffer recipe can be found in sub-section 1.1. Protein concentration is determined by measuring the protein absorption at 280 nm (A280nm).12 The molar extinction coefficient (&#949;) used to determine concentration is &#949; = 1,490 dm3 mol-1 cm-1.13</li>
		<li>Inject 20 &#181;L of the 1 mg/mL (222 &#181;M) solution onto the HPLC column and run the analytical method that was setup in step 2.3. 
		NOTE: The remaining solution not used for analysis can be flash frozen in liquid nitrogen and lyophilized to recover the A&#946; peptide. Lyophilization details can be found in sub-section 1.4.4. The A&#946; peptide will elute from the analytical column between 16 and 18 min (Figure 2D). Use the built-in integration analysis software that accompanies the HPLC instrument to determine the purity of the A&#946; peptide. Purity is determined by integrating each of the individual peaks on the spectrum and calculating peptide-peak percentage area. Typically, a purification of &#62;95% should be ascertained.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/55482/55482fig2.jpg" /> 
Figure 2: Representative HPLC traces of A&#946;42. (A) Traditional C4 silica purification, conditions: Buffer A: H2O with 0.1% trifluoroacetic acid (TFA), buffer B: MeCN (acetonitrile) with 0.1% TFA, gradient: 20 to 27% buffer B over 40 min followed by isocratic 27% buffer B; (B) Preparative purification using the 25 x 300 mm2 polymer column, conditions: Buffer A: H2O with 20 mM NH4OH, buffer B: 80% MeCN / 20% H2O with 20 mM NH4OH, gradient: 20 to 27% buffer B over 70 min followed by isocratic 27% buffer B; (C) Optimized preparative purification using the 25 x 300 mm2 polymer column, conditions are described in Table 1 located in sub-section 1.3 of the protocol description text; (D) Analytical HPLC using the 4.6 x 250 mm2 polymer column, conditions: Buffer A: H2O with 20 mM NH4OH, buffer B: 80% MeCN / 20% H2O with 20 mM NH4OH, gradient-5 to 50% buffer B over 30 min. For parts A, B and C the peak corresponding to A&#946;42 is marked by an asterisk. Collection of the marked A&#946;42 peak in part C reveals a purity of &#62;95% as shown in part D. Mass spectrometry was used to determine the identity of the A&#946;42 peak. <a href="http://cloudfront.jove.com/files/ftp_upload/55482/55482fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<ol>
	<li>Querfurth, H. W., LaFerla, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alzheimer's+Disease.">Alzheimer's Disease.</a> N. Engl. J. Med. 362 (4), 329-344 (2010).</li><li>McGowan, E., 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=A&#946;42+Is+Essential+for+Parenchymal+and+Vascular+Amyloid+Deposition+in+Mice.">A&#946;42 Is Essential for Parenchymal and Vascular Amyloid Deposition in Mice.</a> Neuron. 47 (2), 191-199 (2005).</li><li>Gong, 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=Alzheimer's+disease-affected+brain:+Presence+of+oligomeric+A&#946;+ligands+(ADDLs)+suggests+a+molecular+basis+for+reversible+memory+loss.">Alzheimer's disease-affected brain: Presence of oligomeric A&#946; ligands (ADDLs) suggests a molecular basis for reversible memory loss.</a> Proc. Natl. Acad. Sci. USA. 100 (18), 10417-10422 (2003).</li><li>Selkoe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soluble+Oligomers+of+the+Amyloid+&#946;-Protein+Impair+Synaptic+Plasticity+and+Behavior.">Soluble Oligomers of the Amyloid &#946;-Protein Impair Synaptic Plasticity and Behavior.</a> Behav Brain Res. 192 (1), 106-113 (2008).</li><li>Zagorski, M. G., Yang, J., Shao, H., Ma, K., Zeng, H., Hong, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodological+and+Chemical+Factors+Affecting+Amyloid+&#946;+Peptide+Amyloidogenicity.">Methodological and Chemical Factors Affecting Amyloid &#946; Peptide Amyloidogenicity.</a> Methods Enzymol. 309, 189-204 (1999).</li><li>Kim, W., Hecht, M. 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. J. A., Dutta, S., Foley, A. R., Raskatov, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+of+D-glutamate+at+a+critical+residue+of+A&#946;42+stabilizes+a+pre-fibrillary+aggregate+with+enhanced+toxicity.">Introduction of D-glutamate at a critical residue of A&#946;42 stabilizes a pre-fibrillary aggregate with enhanced toxicity.</a> Chem Eur J. 22 (34), 11967-11970 (2016).</li><li>Thompson, J. A., Lim, T. K., Barrow, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On-line+High-performance+Liquid+Chromatography/Mass+Spectrometric+Investigation+of+Amyloid-&#946;+Peptide+Variants+Found+in+Alzheimer's+Disease.">On-line High-performance Liquid Chromatography/Mass Spectrometric Investigation of Amyloid-&#946; Peptide Variants Found in Alzheimer's Disease.</a> Rapid Commun. 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>

The authors have nothing to disclose.

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</...

Alzheimer&#39;s disease is a neurodegenerative disorder that effects over 35 million people worldwide.1 Implicated strongly in the onset and development of the disease, is the highly aggregation prone, hydrophobic peptide Amyloid beta (A&#946;).2 A&#946; ranges from 36 to 43 amino acids in length, however, it is thought that the 42-amino acid variant, amyloid beta 42 (A&#946;42), is the most toxic form of the protein.3 This is due in most part to the ability of A&#946;42 to readily form diffusible, oligomeric species that are believed to be particularly neurotoxic entities.4 In order to further our understanding of the A&#946; peptide, it is essential to routinely obtain high purity material. The presence of trace impurities has been shown to dramatically alter the aggregation propensity properties of the peptide.5
Traditionally, the high performance liquid chromatography (HPLC) separation of hydrophobic peptides such as A&#946; has been done through the use of a combination of C4 or C8 silica-based stationary phases and an acidic mobile phase.6 However, such conditions can present a challenge to the purification of the peptide. The low isoelectric point of the A&#946; peptide (pI approximately 5.5)7 means that under acidic conditions, peptide aggregation is increased and as a result broad, non-resolved HPLC peaks that are often difficult to isolate are produced (Figure 2A). Furthermore, such broad peaks often contain impurities which may impact the aggregation profile of the peptide, and commonly require subsequent rounds of purification which can dramatically impact the amount of peptide produced.
The poly(styrene/divinylbenzene) stationary phase, PLRP-S, represents an alternative means of purifying hydrophobic peptides. The stationary phase has been employed in the purification of a number of different proteins and messenger ribonucleic acids (mRNA).8,9 The PLRP-S stationary phase requires no additional alkyl ligand for reverse phase separation, and more importantly is chemically stable at high pH which leads to deaggregation of the peptide.7 Herein, we report a tailored HPLC purification protocol that yields high purity amyloid beta 42 (A&#946;42) and amyloid beta 40 (A&#946;40) peptides.

<table border="0" cellpadding="0" cellspacing="0" width="1207">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<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.

The purification of the A&#946;42 peptide using a combination of the PLRP-S stationary phase and a high pH mobile phase results in the formation of a sharp, resolved peak for the A&#946; peptide at a retention time between 72 and 74 min (Figure 2C). Confirmation of the identity of the peak is done through direct injection mass spectrometry of the collected eluent. The eluent can be stored at -20 &#176;C in solution for up to 12 h. Longer periods of storage may result in o...

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

Herein we report a tailored HPLC purification protocol that yields high-purity amyloid beta 42 (A&#946;42) and amyloid beta 40 (A&#946;40) peptides, capable of oligomer formation. Amyloid beta is a highly aggregation prone, hydrophobic peptide implicated in Alzheimer&#39;s disease. The amyloidogenic nature of the peptide makes its purification a challenge.

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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JoVE Journal

Biochemistry

11.7K Views.  University of California, Santa Cruz. 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 we...