Activity-Based Proteomics: Methods and Protocols by Herman S. Overkleeft, Bogdan I. Florea

By Herman S. Overkleeft, Bogdan I. Florea

This quantity specializes in explorative activity-based proteomics,biomedical purposes of activity-based proteomics, and chemical concepts in activity-based proteomics offering a concise assessment of activity-based protein profiling. Written within the hugely profitable Methods in Molecular Biology series structure, chapters contain introductions to their respective themes, lists of the mandatory fabrics and reagents, step by step, effectively reproducible laboratory protocols, and pointers on troubleshooting and warding off identified pitfalls.

Authoritative and state of the art, Activity-Based Proteomics: equipment and Protocols goals to make sure winning ends up in the extra learn of this very important field.

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5. Add 90 μL STSA per 10 μL of your MS samples (see Note 60), vortex briefly and spin down 10 s 16,000 × g. Check with an indicator stick that the pH is around 3. 6. Screw the StageTip holder on a fresh 2 mL tube and place the conditioned StageTip in the hole. Load the peptide solution on the StageTip and centrifuge at 400–800 × g for 2 min (see Note 61). 7. Reapply the flow-through to the StageTip and centrifuge again at 400–800 × g for 2 min (see Note 62). 8. Screw the StageTip holder on a fresh 2 mL tube and place the StageTip with the immobilized peptides in the hole.

The confluent cells are preincubated with either DMSO (Sample A) or only E64-d (Sample B) for 30 min. Subsequently, ≡Sym4 is added to a final concentration of 20 μM and incubated for 6 h. 3. The confluent cells (90 % confluence) are washed 3× with 5 mL sterile 1× DPBS and harvested with a cell scraper in 500 μL sterile 1× DPBS. 4. The cells are pelleted by centrifugation (1000 × g, 4 °C, 5 min) and the supernatant is discarded. In order to lyse the cells they are resuspended in 150 μL lysis buffer and placed on ice for 20 min.

A). Chemical structure of the model bioreactive natural product Symplostatin 4 (Sym4), the alkyne-tagged derivative (≡Sym4) and the trifunctional reporter TriN3. (b). Schematic overview of the steps involved in 2-step ABPP. 1 In Vivo Labeling with Natural Com pound Derivative and Sample Preparation for the Click Chemistry Reaction 1. RPMI 1640 (1×) media (Gibco by Life Technologies, see Note 1): 50 mL heat inactivated FBS (Fetal Bovine Serum; Gibco by Life Technologies) and 5 mL Pen Strep (50,000 Units Penicillin, 50 mg Streptomycin; Gibco by Life Technologies) are added to 500 mL RPMI media.

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