By Brian Vicente, Max Nori, H. Scott Fogler
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The hetero-bifunctional cross-linker LC-SMCC was utilized to couple the small molecule through the free primary amine to a peptide via a cysteine residue. to LC-SMCC, and FLAG® coupled to Bis-III through LC-SMCC, for further purification (see Note 4). 8. 6 mm Kromasil C18 HPLC column with a flow rate of 1 mL/min using an Agilent 1100 HPLC pump. 9. Elute the columns using a linear gradient of water:acetonitrile from 95:5 to 55:65 developed over a period of 45 min with a flow rate of 1 mL/min using a HPLC pump.
Rix et al. 2. Centrifuge the beads for 3 min at room temperature with 75 × g and remove the supernatant. 3. Add 50 μL of DMSO, resuspend gently by inverting several times, centrifuge (as before), and discard the supernatant. 5 mL DMSO. Then resuspend beads in 50 μL DMSO. 4. 75 μL TEA to the 50% bead slurry, mix carefully and incubate on rotoshaker for 16–24 h (but at least for 8 h) at room temperature with 10 rpm (see Note 7). 5. Centrifuge the beads (as before) and remove 10 μL (»5 nmol) of the supernatant for the LC-MS control.
4. Anti-FLAG® M2 affinity resin (Sigma, St. Louis, MO). 5. BioMag® Sheep Anti-Fluorescein IgG (Qiagen, Valencia, CA). 6. Magnetic Separator (Magna-Sep™, Invitrogen, Carlsbad, CA). 4. Sample Preparation for SDS-PAGE and Western Blotting 1. Lab grade chloroform and methanol. 2. LDS sample buffer (Invitrogen, Carlsbad, CA). 3. Reducing Agent (Invitrogen, Carlsbad, CA). 4. 4–12% Bis-Tris precast SDS-PAGE Invitrogen, Carlsbad, CA). gels (NUPAGE®, 5. Mini cell apparatus (XCell Surelock®, Invitrogen, Carlsbad, CA).