Peptide-Based Drug Design

Analysis of Aβ Interactions 81
5. Filter the solution to ensure preformed aggregates are removed (20 �m Minisart
RC4, Sartorius).
6. To account for the loss of peptide resulting from the filtering process, it
is necessary to reassess the protein concentration by UV spectrophotometer.
The extinction coefficient for A� fragments is determined by measuring light
absorbance of A� dissolved in a high-pH solution (e.g., NaOH at pH 11 to allow
for complete solubility).
7. Adjust samples to 10 �M and incubate at 37 ◦ C with shaking (200 rpm) to induce
aggregation.
3.2.2. ProteinChip Analysis
1. Place H50 ProteinChip arrays in a humidity chamber.
2. Pre-equilibrate the arrays in the sample binding buffer; load each spot with 5 �L
of PB and incubate for 5 min on a shaking table. Repeat for a total of two PB
washes.
3. Adjust samples to 10 �M in PB and load 5 �L per spot.
4. Incubate for 2 h at room temperature on a shaking platform.
5. To remove nonspecifically bound peptide, wash spots three times with 5 �LofPB
for 5 min on a shaking platform.
6. Finally wash arrays twice with 1 mM HEPES, pH 7.4 for 1 min.
7. Dry arrays and load 1 �L of 50% CHCA. Allow the array to air-dry and repeat the
CHCA application.
8. Collect data at the low and high mass range.
9. Export data into an Excel spread sheet and determine the signal-to-noise/area
under the curve for each oligomeric species (see Note 11).
3.3. Capture of Aβ on Lipid-Coated ProteinChip Arrays
3.3.1. Vesicle Preparation
1. Combine equal amounts of 1-Palmitoyl-2-oeoyl-sn-glycero-3 [phospho-l-serine]
(POPS) and 1 -palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) (16 mg)
in a 10-mL glass conical flask and dissolve in 2 mL of ethanol-free chloroform.
2. Allow chloroform to evaporate for 1 h under a stream of nitrogen; this will create
a thin layer of lipid in the bottom of the flask.
3. Add acid-washed glass beads and 1 mL of PB to the flask and incubate for 1 h at
37 ◦ C with agitation (220 rpm) to resuspend the lipid layer.
4. Transfer lipid to an Eppendorf tube, sonicate for 15 min, and subject sample to
several freeze–thaw cycles (5×) using liquid nitrogen and a 37 ◦ C water bath.
5. Prepare the extruder apparatus according to the manufacturer’s specifications.
6. Place apparatus on a 37 ◦ C heating block. Pass the lipid through a 0.05 �M presoaked
filter 11 times, after which time the solution will appear more translucent.
7. Dispense the solution into a glass jar and store under nitrogen at 4 ◦ C; use within
48 h.