Recommendations for Peptide Reconstitution:Preparing peptide for neurotoxicity studies, to induce peptide aggregation:The appearance of toxicity has recently been shown to correlate to the extent of beta sheet structure (S. Wang et al. [2001] J. Biol. Chem. 276(45):42027-42034). Recommended preincubation is:1. Dissolve the lyophilized peptide in 0.1% (v/v) trifluoroacetic acid in water at 10 mg/mL
2. Dilute the peptide to 0.5–1.0 mg/mL with PBS (without Ca2+). 1. Incubate at 25°C for 24-48 h (24-36 h is usually sufficient). Neurotoxic activity is usually observed at 30-100ug/ml.
Preparing peptide for studies which require minimal peptide aggregation:1. Dissolve the peptide at a concentration of 1 mg/mL in 100% HFIP (1,1,1,3,3,3-hexafluoro-2-propanol [Sigma-Aldrich, 99.8% ACS reagent grade])
2. Incubate at RT for 2 hours. During the incubation, vortex the peptide solution several times at moderate speed, allowing the HFIP to cover as much of the surface area as possible
3. Dry down the HFIP/peptide solution under a gentle stream of nitrogen gas. Continue drying for an additional 10 minutes. Cap vial immediately
4. Resuspend the peptide in 100% DMSO
5. Incubate the peptide plus DMSO for 12 minutes at RT with periodic vortexing at moderate speed
6. Add 50ul of this DMSO/peptide solution dropwise to 10ml of BSAT-DPBS (see formulation below) while vortexing at moderate speed. Peptides prepared in this manner have been used as standards in ELISA for the detection of beta amyloid in biological samples. When using this peptide in ELISA, it is important that the assay buffer used with the peptide standards has the same composition as the samples under investigation.
