By Marcus Bantscheff (auth.), Gerard Drewes, Marcus Bantscheff (eds.)
The multidisciplinary technology of chemical proteomics experiences how small molecules of man-made or normal starting place bind to proteins and modulate their functionality. In Chemical Proteomics: equipment and Protocols, professional researchers within the box supply key concepts to enquire chemical proteomics concentrating on analytical recommendations, how probes are generated, concepts for the invention of small molecule objectives and the probing of aim functionality, and small molecule ligand and drug discovery. Written within the hugely winning Methods in Molecular Biology™ sequence structure, chapters comprise introductions to their respective subject matters, lists of the mandatory fabrics and reagents, step by step, without difficulty reproducible laboratory protocols, and key pointers on troubleshooting and warding off identified pitfalls.
Authoritative and functional, Chemical Proteomics : tools and Protocols seeks to supply methodologies that may give a contribution to a much wider program of chemical proteomics tools in biochemical and telephone organic laboratories.
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Example text
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).