3 3 cholamidopropyl dimethylammonio 1 propanesulfonate chaps

Bio-Flag-ATM was incubated with varying amounts of MRN, GST-p53 (1–102) peptide, or homeodomain protein, as indicated in figure legends. Proteins were incubated at room temperature for 30 min. Then, 2.5 mL of Dynabeads kilobaseBINDER magnetic streptavidin beads (Invitrogen) were pre-washed with Buffer A containing 100 mM NaCl and 1 mg/mL BSA and incubated with the protein mixture for 15 min at 4°C in the presence of 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) (Sigma). Biotinylated protein and associated factors were isolated using a magnet and were washed three times with Buffer A containing 100 mM NaCl and 0.1% CHAPS. Beads were resuspended in 2.53 SDS loading buffer and boiled for 10 min before loading onto a 9% denaturing SDS-PAGE gel. Proteins were transferred for 3 hr at 400 mA and analyzed by western blotting as indicated in figure legends.

Soluble biotinylated CD1b monomers were provided by the National Institutes of Health Tetramer Core Facility (Emory University, Atlanta, GA). The loading protocol for CD1b monomers was based on previously published glucose monomycolate loading protocols (Kasmar et al., 2011 (link)). Natural Ac₂SGL was dried down in a glass tube in a stream of nitrogen and sonicated into a 50 mM sodium citrate buffer at pH 4, containing 0.25% with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) (Sigma, St. Louis, MO) for two minutes at 37°C. The sonicate was transferred to a microfuge tube, and 20 μg of CD1b monomer was added and incubated in a 37°C water bath for 2 hours with vortexing every 30 minutes. At the end of the incubation, the solution was neutralized to pH 7.4 with 6 μl of 1M Tris pH 9. For SL37 Ac₂SGL and AM Ac₂SGL, the sonication was performed in 50 mM sodium citrate buffer at pH 7.4, containing 10 mM taurocholate (Sigma, St. Louis, MO) for 30 minutes at 37°C. After addition of CD1b, the mixture was incubated in a 37°C water bath for 2 hours. Finally, 10 μl of Streptavidin conjugated to allophycocyanin or phycoerythrin (Life Technologies, Carlsbad, CA) was added in ten aliquots of 1 μl every 10 minutes. The final product was filtered through a SpinX column (Sigma, St. Louis, MO) to remove aggregates and stored at 4°C until use.

The spray solvent used for MS acquisition was methanol, purchased from Mallinckrodt Baker Inc. (Phillipsburg, NJ), and applied manually via an adjustable pipette (Eppendorf Research-2.5 μL). Additional spray additives including 3-[(3-Cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) was purchased from Sigma-Aldrich (St. Louis, MO). Therapeutic drugs were acquired from Cerilliant (Round Rock, Texas, USA). Drugs were stored at −20° C until diluted in bovine blood to appropriate concentrations. Illicit drugs were acquired from Cerilliant (Round Rock, Texas, USA). Drugs were stored at −20° C until diluted to appropriate concentrations. Non-organic orange was purchased from national grocer for agrochemical MS experiments.