By Herman S. Overkleeft, Bogdan I. Florea
This quantity specializes in explorative activity-based proteomics,biomedical functions of activity-based proteomics, and chemical options in activity-based proteomics offering a concise review of activity-based protein profiling. Written within the hugely profitable Methods in Molecular Biology series layout, chapters comprise introductions to their respective themes, lists of the required fabrics and reagents, step by step, with ease reproducible laboratory protocols, and tips about troubleshooting and heading off recognized pitfalls.
Authoritative and state-of-the-art, Activity-Based Proteomics: equipment and Protocols goals to make sure winning ends up in the additional examine of this important field.
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Extra resources for Activity-Based Proteomics: Methods and Protocols
T-butanol–DMSO solution (t-BuOH:DMSO 1:4): Place the bottle with t-butanol (Sigma, 360538) in a water bath set to 40 °C. Over the next 20 min, agitate the bottle several times to help the melting process. Meanwhile, add 40 mL DMSO to a 50 mL Falcon tube. When all t-butanol has melted, transfer 10 mL of the liquid with a preheated 10 mL glass pipette (see Note 3) to the 50 mL DMSO-containing Falcon tube. Close the Falcon tube and mix by inverting the tube several times. Store the solution at room temperature.
When you hand in your sample for LC-MS, please indicate the composition of your LCSS to the MS operator. 19. Use whatever assay is established in your lab and is compatible with SDS and Triton X-100. Expect 4-7 μg/μL. 20. 5 mL final volume. Make sure that your reaction vessel can accommodate at least 3 mL. 2). 21. You may also use 1 % SDS to stop the reaction, denature the proteins and keep proteins in solution in the subsequent steps. 3). Activity-Based Protein Profiling with Natural Product-Derived Chemical Probes… 39 22.
Transfer the solution to a graduated glass cylinder and adjust the volume to 250 mL with MS water. 22 μm filter unit. The solution can be stored at RT for several months. 3 Affinity Purification 1. Disposable PD-10 desalting columns (GE Healthcare). 2. 1× PBS: Mix 10 mL 10× PBS solution with 90 mL ddH2O. 3. 22 μm filter unit attached to a syringe. 4. Sodium dodecyl sulfate (10 % w/v SDS): 350 mM SDS. 1. 5. Equilibrated Avidin beads from Sigma Aldrich (see Note 5): We usually use 100 μL of the 50 % slurry provided by the manufacturer per 1 mg protein or reaction.
Activity-Based Proteomics: Methods and Protocols by Herman S. Overkleeft, Bogdan I. Florea