Materials, Instrument and Methods
Materials: The CE-SDS MW kit (PN 390953) and LIF Performance Test Mix (PN: 726022) were from SCIEX, Framingham, MA. Packaged AAV8 of pAV-CMV-GFP with titer at 1.10x1013 GC/mL (titer as supplied by vendor) was purchased from Vigene Biosciences (Rockville, MD, U.S.A.). AAV formulation buffer (1X PBS with 0.001% Pluronic F68) was also from Vigene Biosciences. Chromeo P503 catalog # 15106, was purchased from Active Motif (Carlsbad, CA). Phosphate Buffered Saline Bioreagent Suitable for Cell Culture 10X, Sigma-Aldrich, PN P5493-1L was used as base for CE-SDS custom sample buffer. Sodium Dodecyl Sulfate, J. T. Baker, PN 4095-04.
Sample storage: Upon arrival, 5 µL aliquot of AAV8 sample were stored at -80oC freezer to avoid multiple freeze-thaw cycles.
Instrument and software: A PA 800 Plus Pharmaceutical Analysis System equipped with LIF detector and solid-state laser with excitation wavelength at 488 nm were from SCIEX (Framingham, MA) and a 600 nm band pass emission filter from Edmund Optics (Barrington, NJ). Data acquisition and analysis were performed using 32 Karat softwareTM V10. The separation method used in this work has already been described by Li.4 Briefly, the CE separation method takes advantage of stacking technique by introducing a plug of water (20 psi/0.6 min) prior to the sample injections (-10 kV/60 seconds).
LIF Calibration: To ensure consistent response of LIF detector throughout this study, the LIF detector was calibrated using LIF Calibration Wizard and Performance Test Mix (PN: 726022).
Preparation of Chromeo P503 Working Solution: A vial of Chromeo P503 dye comes in 1 mg of lyophilized powder. The lyophilized powder was reconstituted by adding 1 mL of methanol. Make 10 µL aliquots to prevent contamination due to over-handling. After reconstitution, the dye label can be stored at 2-8 oC for six months according to the manufacturer’s instructions.
Sample Dilution Procedure Prior to Labeling: One 5 µL aliquot of AAV8 sample was taken out of the freezer and diluted as follows: To prepare 1.10x1011 GC/mL: 1 microliter of AAV8 1.10x1013 GC/mL was added to a 99 µL of 50 mM phosphate buffer solution pH 8. To prepare 1.10x1010 GC/mL: 1 microliter of AAV8 1.10x1013 GC/mL was added to a 9 µL of 50 mM phosphate buffer solution pH 8.
Sample Denaturing Procedure Prior to Labeling: 5 µL of AAV8 diluted as in previous session were mixed with 5 µL of Tris sample buffer and 1µL of 1M DTT. Both reaction mixes were briefly vortexed for proper mixing and heated to 60 oC for 10 minutes. After, the reaction tubes were allowed to cool down to room temperature.
Sample Labeling Procedure: In this protocol, the least amount of sample possible is used, which may lead to challenges in pipetting very small volume. Scaling up the sample prep is possible and should be used if sample volume is not an issue. 0.5 µL of 1 mg/mL of Chromeo P503 Labeling Working Solution was added to each reaction tube and briefly vortexed. Once again, both tubes were heated to 60 oC for 20 minutes. Afterwards, both tubes were first allowed to cool to room temperature and then 38.5 µL of DDI water was added to either reaction tube. After labeling and dilution, the final concentrations are AAV8 1.1X1010 and 1.1X109 GC/mL respectively.