By Herman S. Overkleeft, Bogdan I. Florea
This quantity specializes in explorative activity-based proteomics,biomedical functions of activity-based proteomics, and chemical concepts in activity-based proteomics offering a concise assessment of activity-based protein profiling. Written within the hugely winning Methods in Molecular Biology series structure, chapters comprise introductions to their respective themes, lists of the required fabrics and reagents, step by step, simply reproducible laboratory protocols, and pointers on troubleshooting and averting identified pitfalls.
Authoritative and state of the art, Activity-Based Proteomics: equipment and Protocols goals to make sure profitable leads to the extra learn of this important field.
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Extra resources for Activity-Based Proteomics: Methods and Protocols
Once the labeling reaction has been stopped by addition of urea, check the pH of the reaction solution. Use an indicator stick. The pH should be between 7 and 8 (see Note 22). Activity-Based Protein Profiling with Natural Product-Derived Chemical Probes… 31 2. Add 20 μL of the 1 mM TriN3 reporter (final concentration should be around ~20 μM) to each of the sample vials and vortex briefly. 3. Add 10 μL of the 100 mM TCEP solution (final concentration in this case is ~1 mM) and vortex briefly (see Note 23).
Denaturing SDS Polyacrylamide gel (for protein separation): this can be either self-made or commercial. The percentage of 28 Susanne Zweerink et al. acrylamide depends on the molecular weight of the protein of interest. We recommend to generally use SDS gels with 10–13 % acrylamide. Lower percentage gels are hard to handle, higher percentage gels cause problems during the in-gel digestion. We prefer to make our own gels. We cast the gels between low fluorescent glass plates which facilitates detection of fluorescence in the following step.
64. You can also increase the volume to 100 μL in the beginning. In extractions, it is important to use two small volumes (thereby following Nernst’s distribution law). In this case, however, we are continuously eluting from the column; thus, it is not really relevant if you use twice 50 μL or once 100 μL. 65. The prepared LC/MS samples are ready for MS now. They can be kept at 4 °C for several days or almost indefinitely frozen at −20 °C. g. with LC-MS sample solution. 5 Discussion The described protocol is robust and allows the routine identification of natural products when an alkyne (or azide) derivative is available.