Peptide-Based Drug Design

Analysis of Aβ Interactions 83
2. Incubate the samples for 5 min with agitation to allow the peptide to bind to the
lipid layer (see Note 12).
3. To remove nonspecifically bound protein, wash arrays twice with PB for 2 min,
followed by two 1-min washes with 1 mM HEPES, pH 7.2.
4. Allow the arrays to air-dry for approx 10 min and load l �L of 50% CHCA. Once
the arrays are dry, load another application of l �L of 50% CHCA and dry again.
5. Collect data at low and high molecular weight range.
6. Export data into an Excel spread sheet and determine the lipid binding affinity of
the oligomers by assessing the signal-to-noise/area under the curve (see Note 11).
7. See Fig. 3 for a summary of the A� lipid-binding assay.
4. Notes
1. It is advantageous to initially screen all the available chip types, including PS10,
PS20, RS100, and PG20, to determine which array provides the highest degree
of sensitivity.
2. To assess the purity and quality of the Ab intended for use on preactivated arrays,
screen the Ab on NP20 arrays (normal phase arrays); also refer to the product
data sheet for the presence of carrier proteins such as BSA and gelatin.
3. The binding affinity of Protein G to Ab is species and isotype dependent; rabbit,
goat, and bovine pAb all exhibit high affinity and hence are the preferred Ab
for PG20 arrays. Mouse monoclonal Ab (mAb) IgG1, a common mAb isotype,
exhibits low binding affinity to Protein G and is not recommended.
4. Allow the binding interaction to reach equilibrium (minimum of 30–60 min).
This will minimize differences in the intensity and peak number among the replicates,
improving reproducibility.
5. If nonspecific binding is obscuring A� detection, consider increasing the (1)
wash time, (2) number of washes, (3) wash volume by using a bioprocessor, or
(4) concentration of detergent/salts. Alternatively, detergent/salt can be added to
the sample during the Ag-binding step.
6. A final 1mM HEPES, pH 7.2 wash is performed to remove contaminants such as
salts and detergent, which may interfere with interpretation of spectra. Formation
of salt adducts creates additional peaks with a mass shift of 22 and 38 Da
for sodium and potassium, respectively, which can affect the sensitivity of the
assay. Detergents generate a series of low molecular weight peaks, which can
complicate analysis and decrease the specific signal.
7. Do not allow extended drying time (>20 min) as this can significantly reduce the
reproducibility of the assay.
8. As EAM addition requires working with low volumes, it is critical to use a
well-calibrated 2-�L pipet. Further to this, it is essential to be consistent as the
application of EAM and drying time affect crystallization and hence the signal
intensity.
9. It is advantageous to have two data acquisition settings: one for the low molecular
weight range (0–20 kDa) and the other for the high molecular weight range