By Marcus Bantscheff (auth.), Gerard Drewes, Marcus Bantscheff (eds.)
The multidisciplinary technological know-how of chemical proteomics stories how small molecules of artificial or ordinary starting place bind to proteins and modulate their functionality. In Chemical Proteomics: equipment and Protocols, specialist researchers within the box supply key options to enquire chemical proteomics targeting analytical thoughts, how probes are generated, innovations for the invention of small molecule goals and the probing of aim functionality, and small molecule ligand and drug discovery. Written within the hugely winning Methods in Molecular Biology™ sequence layout, chapters comprise introductions to their respective issues, lists of the mandatory fabrics and reagents, step by step, without problems reproducible laboratory protocols, and key tips about troubleshooting and heading off identified pitfalls.
Authoritative and useful, Chemical Proteomics : equipment and Protocols seeks to supply methodologies that would give a contribution to a much wider program of chemical proteomics tools in biochemical and mobile organic laboratories.
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Additional info for Chemical Proteomics: Methods and Protocols
It is therefore of critical importance to define the cellular target proteins of a compound as an entry point to understanding its mechanism of action. Often, this can be achieved in a direct fashion by chemical proteomics. As with any affinity chromatography, immobilization of the bait to a solid support is one of the earliest and most crucial steps in the process. Interfering with structural features that are important for identification of a target protein will be detrimental to binding affinity.
5. γ-Globin (1 μg/μL) (Bio-Rad). 6. Protein Assay Dye Reagent Concentrate (5×) (Bio-Rad). 7. Hypodermic Needle, 20 G × 1½ (Terumo®), single-use syringe 10 mL (B. Braun Melsungen AG). 8. 5 mL, PMMA (VWR International). 9. Polycarbonate ultracentrifuge tubes (Beckman Coulter). 10. Ultracentrifuge and fixed-angle rotor (Beckman Coulter). 28 U. Rix et al. 4. Affinity Pulldown 1. Lysis buffer 1× (see above). 2. 4× Concentrated Laemmli sample buffer (containing 10% β-mercaptoethanol) (see above). 3. Polycarbonate ultracentrifuge tubes (Beckman Coulter).
0 × 50 mm (Waters). 6. HPLC 2795 (Waters). 7. Photo diode array detector 2996 (Waters). 8. ZQ 2000 single quadrupole mass spectrometer (Waters/ Micromass). 3. Cell Culture, Harvest, Lysis, and Protein Quantification 1. Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (Gibco) and penicillin/ streptomycin 100× (PAA Laboratories GmbH). 2. Cell culture dishes, Ø 150 mm (VWR International). 3. 4× Concentrated Laemmli sample buffer containing 10% β-mercaptoethanol.
Chemical Proteomics: Methods and Protocols by Marcus Bantscheff (auth.), Gerard Drewes, Marcus Bantscheff (eds.)