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="https://cloudfront-jove-com.remotexs.ntu.edu.sg/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="https://cloudfront-jove-com.remotexs.ntu.edu.sg/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).
To circumvent this issue, the PLRP-S stationary phase, chemically stable at high pH was found to be effective for the purification of the A&#946; peptide (Figure 2B and 2C). The use of a high pH mobile phase minimized the degree of aggregation, and when optimized, gave rise to a sharp, well-resolved peak. In the optimized protocol, the percentage of buffer B was rapidly switched from 20% to 27% at the 52-minute time point and as a result, gave rise to a well-defined A&#946; peak (Figure 2C). This optimized protocol relies on all of the hydrophobic impurities being eluted from the column during the initial 20 to 24.5% increase in buffer B concentration. If impurities are found which are of a similar hydrophobicity as the A&#946; peptide, then further optimization of the HPLC protocol may be warranted. The optimized method was highly reproducible amongst individual batches of synthesized A&#946; and was capable of purifying both 40 and 42 variants of the peptide without any changes to the optimized procedure (Figure 3). For both peptides, the purity as determined by analytical HPLC was found to be greater than 95%.
Given the reports of trace impurities altering the aggregation propensity of the A&#946; peptide, it is advisable that an orthogonal biophysical characterization be employed to confirm that the purified peptide is able to undergo oligomerization. Using previously reported protocols we chose to perform PICUP.14,15 PICUP analysis of the purified proteins demonstrates the characteristic monomer through hexamer population distribution for the A&#946;40 peptide and the monomer through heptamer distribution associated with the A&#946;42 peptide.10 These results confirm that purification of the A&#946; peptides using the PLRP-S stationary phase and a high pH mobile eluent results in A&#946; proteins that are capable of aggregation.
The purification procedure reported is designed to be able to purify up to 3 mg of the A&#946; peptide in a single chromatographic run. For this procedure, it is critical to set the flow rate of the HPLC instrument at 6 mL/min. If lower flow rates of the HPLC pump are used, the HPLC run time should be extended to accommodate this. Conversely, the use of higher flow rates may warrant a reduction in the HPLC run time. However, reduction of the run time can result in the co-elution of the A&#946; peak with other peaks and therefore lower the purity of the A&#946; peptide collected. Furthermore, the HPLC instrumentation and size of the chromatographic column used for purification can greatly affect the amount of material that can be purified in a single run. If no peak is produced at the expected eluent time of the A&#946; peptide, and a large peak is produced at the eluent time corresponding to the solvent front, then too much material was initially loaded onto the column. Reducing the amount of material injected onto the HPLC column for each run will circumvent this problem. Once the A&#946; peptide has been purified, it is highly recommended that the solution be lyophilized as quickly as possible. Prolonged storage of the peptide in solution can lead to oxidation of A&#946; and therefore formation of impurities. The purity of the A&#946; peptide should always be determined by analytical HPLC. Trace amounts of impurities can alter the aggregation propensity of the peptide dramatically. If the analytical HPLC trace shows the presence of impurities, then the sample should be re-subjected to the HPLC purification protocol and its purity re-determined by analytical HPLC.
It is hoped that this tailored procedure for the purification of the A&#946;42 and A&#946;40 peptides will be of benefit for the scientific community and allow users to obtain high purity A&#946; capable of oligomer formation. It would be expected that this procedure could be adapted to other amyloidogenic peptides that are difficult to isolate and purify.

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 oxidation of the protein. To isolate the purified peptide, the eluent is flash frozen in liquid nitrogen and lyophilized. Analytical HPLC of the purified peptide reveals a purity of &#62;95% (Figure 2D). The same procedure can be used to purify the A&#946;40 peptide (Figure 3A) without modification of the method. Analytical HPLC indicates a purity of A&#946;40 to be greater than 95% (Figure 3B).
<img alt="Figure 3" src="/files/ftp_upload/55482/55482fig3.jpg" /> 
Figure 3: Representative HPLC traces of A&#946;40. (A) 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. The peak corresponding to A&#946;40 is marked with an asterisk. (B) 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. Collection of the marked A&#946;40 peak in part A reveals a purity of &#62;95% as shown in part B. Mass spectrometry was used to determine the identity of the A&#946;40 peak. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55482/55482fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In order to confirm that the purified peptide can form oligomeric mixtures, we performed photo-induced crosslinking of unmodified proteins (PICUP) as previously described14,15 and were able to robustly observe oligomer formation.10

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

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 ...

a-tailored-hplc-purification-protocol-that-yields-high-purity-amyloid

Nanyang Technological University

Research

JoVE Journal

Biochemistry

11.8K Views.  University of California, Santa Cruz. Herein we report a tailored HPLC purification protocol that yields high-purity amyloid beta 42 (Aβ42) and amyloid beta 40 (Aβ40) peptides, capable of oligomer formation. Amyloid beta is a highly aggregation prone, hydrophobic peptide implicated in Alzheimer's disease. The amyloidogenic nature of the peptide makes its purification a challenge.

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

Laboratory Scale Production and Purification of a Therapeutic Antibody

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the antibody genes followed by stable transfection into a suitable cell line. The stable clones are subjected to screening in order to select those clones with desired production and growth characteristics. This is a critical albeit time-consuming step in the process. This protocol considers vector selection and sourcing of antibody sequences for the expression of a therapeutic antibody. The methods describe preparation of vector DNA for stable transfection of a suspension variant of human embryonic kidney 293 (HEK-293) cell line, using polyethylenimine (PEI). The cells are transfected as adherent cells in serum-containing media to maximize transfection efficiency, and afterwards adapted to serum-free conditions. Large scale production, setup as batch overgrow cultures is used to yield antibody protein that is purified by affinity chromatography using an automated fast protein liquid chromatography (FPLC) instrument. The antibody yields produced by this method can provide sufficient protein to begin initial characterization of the antibody. This may include in vitro assay development or physicochemical characterization to aid in the time-consuming task of clonal screening for lead candidates. This method can be transferable to the development of an expression system for the production of biosimilar antibodies.

This protocol describes the production of a therapeutic antibody in a mammalian expression system. The methods described include preparation of vector DNA, stable transfection and serum-free adaptation of a human embryonic kidney 293 cell line, set up of large scale cultures and purification using affinity chromatography.

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the ...

Expression, Purification, and Antimicrobial Activity of S100A12

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some S100 proteins have the capacity to bind transition metals with high affinity and effectively sequester them away from invading microbial pathogens in a process that is termed "nutritional immunity". S100A12 (EN-RAGE) binds both zinc and copper and is highly abundant in innate immune cells such as macrophages and neutrophils. We report a refined method for the expression, enrichment and purification of S100A12 in its active, metal-binding configuration. Utilization of this protein in bacterial growth and viability analyses reveals that S100A12 has antimicrobial activity against the bacterial pathogen, Helicobacter pylori. The antimicrobial activity is predicated on the zinc-binding activity of S100A12, which chelates nutrient zinc, thereby starving H. pylori which requires zinc for growth and proliferation.

Here, we present a method to express and purify S100A12 (calgranulin C). We describe a protocol to measure its antimicrobial activity against the human pathogen H. pylori.

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some ...

Imaging Amyloid Tissues Stained with Luminescent Conjugated Oligothiophenes by Hyperspectral Confocal Microscopy and Fluorescence Lifetime Imaging

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find neurodegenerative diseases such as Alzheimer&#39;s and Parkinson&#39;s disease afflicting primarily the central nervous system, and systemic amyloidosis where serum amyloid A, transthyretin and IgG light chains deposit as amyloid in liver, carpal tunnel, spleen, kidney, heart, and other peripheral tissues. Amyloid has been known and studied for more than a century, often using amyloid specific dyes such as Congo red and Thioflavin T (ThT) or Thioflavin (ThS). In this paper, we present heptamer-formyl thiophene acetic acid (hFTAA) as an example of recently developed complements to these dyes called luminescent conjugated oligothiophenes (LCOs). hFTAA is easy to use and is compatible with co-staining in immunofluorescence or with other cellular markers. Extensive research has proven that hFTAA detects a wider range of disease associated protein aggregates than conventional amyloid dyes. In addition, hFTAA can also be applied for optical assignment of distinct aggregated morphotypes to allow studies of amyloid fibril polymorphism. While the imaging methodology applied is optional, we here demonstrate hyperspectral imaging (HIS), laser scanning confocal microscopy and fluorescence lifetime imaging (FLIM). These examples show some of the imaging techniques where LCOs can be used as tools to gain more detailed knowledge of the formation and structural properties of amyloids. An important limitation to the technique is, as for all conventional optical microscopy techniques, the requirement for microscopic size of aggregates to allow detection. Furthermore, the aggregate should comprise a repetitive &#946;-sheet structure to allow for hFTAA binding. Excessive fixation and/or epitope exposure that modify the aggregate structure or conformation can render poor hFTAA binding and hence pose limitations to accurate imaging.

Amyloid deposition is a hallmark of different diseases and afflicts many different organs. This paper describes the application of luminescent conjugated oligothiophene fluorescence staining in combination with fluorescence microscopy techniques. This staining method represents a powerful tool for detection and exploration of protein aggregates in both clinical and scientific setups.

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find ...

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be greatly facilitated by the production of milligram amounts of pure, folded ligand binding domains. Attempts to heterologously express periplasmic ligand binding domains of bacterial chemoreceptors in Escherichia coli (E. coli) often result in their targeting into inclusion bodies. Here, a method is presented for protein recovery from inclusion bodies, its refolding and purification, using the periplasmic dCACHE ligand binding domain of Campylobacter jejuni (C. jejuni) chemoreceptor Tlp3 as an example. The approach involves expression of the protein of interest with a cleavable His6-tag, isolation and urea-mediated solubilisation of inclusion bodies, protein refolding by urea depletion, and purification by means of affinity chromatography, followed by tag removal and size-exclusion chromatography. The circular dichroism spectroscopy is used to confirm the folded state of the pure protein. It has been demonstrated that this protocol is generally useful for production of milligram amounts of dCACHE periplasmic ligand binding domains of other bacterial chemoreceptors in a soluble and crystallisable form.

A procedure is presented for the refolding of the dCACHE periplasmic ligand binding domain of Campylobacter jejuni chemoreceptor Tlp3 from inclusion bodies and the purification to yield milligram quantities of protein.

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be ...

Use of Two Dimensional Semi-denaturing Detergent Agarose Gel Electrophoresis to Confirm Size Heterogeneity of Amyloid or Amyloid-like Fibers

Amyloid or amyloid-like fibers have been associated with many human diseases, and are now being discovered to be important for many signaling pathways. The ability to readily detect the formation of these fibers under various experimental conditions is essential for understanding their potential function. Many methods have been used to detect the fibers, but not without some drawbacks. For example, electron microscopy (EM), or staining with Congo Red or Thioflavin T often requires purification of the fibers. On the other hand, semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) allows detection of the SDS-resistant amyloid-like fibers in the cell extracts without purification. In addition, it allows the comparison of the size difference of the fibers. More importantly, it can be used to identify specific proteins within the fibers by Western blotting. It is less time consuming and more easily accessible to a wider number of labs. SDD-AGE results often show variable degree of heterogeneity. It raises the question whether part of the heterogeneity results from the dissociation of the protein complex during the electrophoresis in the presence of SDS. For this reason, we have employed a second dimension of SDD-AGE to determine if the size heterogeneity seen in SDD-AGE is truly a result of fiber heterogeneity in vivo and not a result of either degradation or dissociation of some of the proteins during electrophoresis. This method allows fast, qualitative confirmation that the amyloid or amyloid-like fibers are not partially dissociating during the SDD-AGE process.

Here we use two-dimensional semi-denaturing agarose gel electrophoresis to confirm the presence of amyloid-like fibers of heterogeneous size and exclude the possibility that the size heterogeneity is due to dissociation of the amyloid fibers during the gel running process.

Amyloid or amyloid-like fibers have been associated with many human diseases, and are now being discovered to be important for many signaling pathways. ...

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation of protein synthesis is the key to cellular stress-response, and dysregulation is central to many disease states, such as cancer. For instance, although cellular stress leads to the inhibition of global translation by attenuating cap-dependent initiation, certain stress-response proteins are selectively translated in a cap-independent manner. Discreet RNA regulatory elements, such as cellular internal ribosome entry sites (IRESes), allow for the translation of these specific mRNAs. Identification of such mRNAs, and the characterization of their regulatory mechanisms, have been a key area in molecular biology. Toeprinting is a method for the study of RNA structure and function as it pertains to translation initiation. The goal of toeprinting is to assess the ability of in vitro transcribed RNA to form stable complexes with ribosomes under a variety of conditions, in order to determine which sequences, structural elements, or accessory factors are involved in ribosome binding&#8212;a pre-cursor for efficient translation initiation. Alongside other techniques, such as western analysis and polysome profiling, toeprinting allows for a robust characterization of mechanisms for the regulation of translation initiation.

Toeprinting aims to measure the ability of in vitro transcribed RNA to form translation initiation complexes with ribosomes under a variety of conditions. This protocol describes a method for toeprinting mammalian RNA and can be used to study both cap-dependent and IRES-driven translation.

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation ...

A11-positive &#946;-amyloid Oligomer Preparation and Assessment Using Dot Blotting Analysis

&#946;-amyloid (A&#946;) is a hydrophobic peptide with an intrinsic tendency to self-assemble into aggregates. Among various aggregates, A&#946; oligomer is widely accepted as the leading neurotoxin in the progress of Alzheimer&#39;s disease (AD) and is considered to be the crucial event in the pathogenesis of AD. Therefore, A&#946; oligomer inhibitors might prevent neurodegeneration and have the potential to be developed as disease-modifying treatments of AD. However, different formation protocols of A&#946; oligomer might lead to oligomers with different characteristics. Moreover, there are not many methods to effectively screen A&#946;1-42 oligomer inhibitors. An A11 antibody can react with a subset of toxic A&#946;1-42 oligomer with anti-parallel &#946;-sheet structures. In this protocol, we describe how to prepare an A11-positive A&#946;1-42 oligomer-rich sample from a synthetic A&#946;1-42 peptide in vitro and to evaluate relative amounts of A11-positive A&#946;1-42 oligomer in samples by a dot blotting analysis using A11 and A&#946;1-42-specific 6E10 antibodies. Using this protocol, inhibitors of A11-positive A&#946;1-42 oligomer can also be screened from semi-quantitative experimental results.

This protocol describes how to prepare A&#946; oligomers from a synthetic peptide in vitro and to evaluate relative amounts of A&#946; oligomer by a dot blotting analysis.

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

Purification and Reconstitution of TRPV1 for Spectroscopic Analysis

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine and remain largely unknown. With recent advances in single-particle cryo-electron microscopy (cryo-EM) shedding light on the structural features of agonist binding sites and the activation mechanism of several ion channels, the stage is set for an in-depth dynamic analysis of their gating mechanisms using spectroscopic approaches. Spectroscopic techniques such as electron paramagnetic resonance (EPR) and double electron-electron resonance (DEER) have been mainly restricted to the study of prokaryotic ion channels that can be purified in large quantities. The requirement for large amounts of functional and stable membrane proteins has hampered the study of mammalian ion channels using these approaches. EPR and DEER offer many advantages, including determination of the structure and dynamic changes of mobile protein regions, albeit at low resolution, that might be difficult to obtain by X-ray crystallography or cryo-EM, and monitoring reversible gating transition (i.e., closed, open, sensitized, and desensitized). Here, we provide protocols for obtaining milligrams of functional detergent-solubilized transient receptor potential cation channel subfamily V member 1 (TRPV1) that can be labeled for EPR and DEER spectroscopy.

This article describes specific methods to obtain biochemical quantities of detergent-solubilized TRPV1 for spectroscopic analysis. The combined protocols provide biochemical and biophysical tools that can be adapted to facilitate structural and functional studies for mammalian ion channels in a membrane-controlled environment.

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

Detection and Isolation of Apoptotic Bodies to High Purity

Apoptotic bodies (ApoBDs), microvesicles and exosomes are the key members of the extracellular vesicle family, with ApoBDs being one of the largest type. It has been proposed that ApoBDs can aid cell clearance as well as intercellular communication through trafficking biomolecules. Conventional approaches used for the identification and isolation of ApoBDs are often limited by the lack of accurate quantification and low sample purity. Here, we describe a workflow to confirm the induction of apoptosis, validate ApoBD formation, and isolate ApoBDs to high purity. We will also outline and compare fluorescence-activated cell sorting (FACS) and differential centrifugation based approaches to isolate ApoBDs. Furthermore, the purity of isolated ApoBDs will be confirmed using a previously establish flow cytometry-based staining and analytical method. Taken together, using the described approach, THP-1 monocyte apoptosis and apoptotic cell disassembly was induced and validated, and ApoBD generated from THP-1 monocytes were isolated to a purity of 97-99%.

A workflow using flow cytometry or differential centrifugation is developed to detect, quantify and isolate apoptotic bodies from an apoptotic sample to high purity.

Apoptotic bodies (ApoBDs), microvesicles and exosomes are the key members of the extracellular vesicle family, with ApoBDs being one of the largest type. ...

Analysis of &#946;-Amyloid-induced Abnormalities on Fibrin Clot Structure by Spectroscopy and Scanning Electron Microscopy

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A&#946;) protein on clot formation and structure by spectrometry and scanning electron microscopy (SEM). A&#946;, which forms neurotoxic amyloid aggregates in Alzheimer&#39;s disease (AD), has been shown to interact with fibrinogen. This A&#946;-fibrinogen interaction makes the fibrin clot structurally abnormal and resistant to fibrinolysis. A&#946;-induced abnormalities in fibrin clotting may also contribute to cerebrovascular aspects of the AD pathology such as microinfarcts, inflammation, as well as, cerebral amyloid angiopathy (CAA). Given the potentially critical role of neurovascular deficits in AD pathology, developing compounds which can inhibit or lessen the A&#946;-fibrinogen interaction has promising therapeutic value. In vitro methods by which fibrin clot formation can be easily and systematically assessed are potentially useful tools for developing therapeutic compounds. Presented here is an optimized protocol for in vitro generation of the fibrin clot, as well as analysis of the effect of A&#946; and A&#946;-fibrinogen interaction inhibitors. The clot turbidity assay is rapid, highly reproducible and can be used to test multiple conditions simultaneously, allowing for the screening of large numbers of A&#946;-fibrinogen inhibitors. Hit compounds from this screening can be further evaluated for their ability to ameliorate A&#946;-induced structural abnormalities of the fibrin clot architecture using SEM. The effectiveness of these optimized protocols is demonstrated here using TDI-2760, a recently identified A&#946;-fibrinogen interaction inhibitor.

Presented here are two methods that can be used individually or in combination to analyze the effect of beta-amyloid on fibrin clot structure. Included is a protocol for creating an in vitro fibrin clot, followed by clot turbidity and scanning electron microscopy methods.

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A&#946;) protein on clot formation and ...

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

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