Peptide-Based Drug Design

Analysis of Aβ Interactions 79
4. Centrifuge for 1 min at 13,000 rpm and combine 100 �L of 100% saturated CHCA
with 100 �L of 50% ACN, 0.5% TFA to make 200 �L of 50% saturated CHCA.
Prepare CHCA in an opaque tube as it is light sensitive.
5. Load 1 �L of 50% CHCA to each spot and allow to air-dry. Repeat this step,
ensuring spots are dry before addition of the second layer of CHCA (see Note 8).
6. See Fig. 2 for a summary of the A� capture assay.
3.1.2.5. DATA ACQUISITION SETTINGS
Laser and sensitivity settings need to be optimized for each new set of experimental
conditions. Excess laser energy will generate flat-topped broad peaks
Fig. 2. Capture of A� using Ab-coated ProteinChip arrays. Preactivated arrays with
exposed active sites are shown in (A). Ab are covalently bound to the array via amine
groups (B). Exposed active sites are blocked using Tris-containing buffer (C). Samples
are added to the array, incubated for 1–4 h (D), and washed to remove nonspecifically
bound proteins (E). EAM is applied to each spot (F), and the array is introduced into the
ProteinChip reader where the laser is fired onto the chip surface. Proteins retained on
the array surface are subsequently resolved via TOF-MS, which displays the mass-tocharge
value and signal intensities of each protein (G, H). (Adapted from ProteinChip
technology training course, Bio-Rad Laboratories.)