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

By Herman S. Overkleeft, Bogdan I. Florea

This quantity makes a speciality of explorative activity-based proteomics,biomedical purposes of activity-based proteomics, and chemical innovations in activity-based proteomics offering a concise assessment of activity-based protein profiling. Written within the hugely profitable Methods in Molecular Biology series layout, chapters contain introductions to their respective subject matters, lists of the mandatory fabrics and reagents, step by step, comfortably reproducible laboratory protocols, and tips about troubleshooting and averting recognized pitfalls.

Authoritative and state-of-the-art, Activity-Based Proteomics: tools and Protocols goals to make sure profitable leads to the extra examine of this important field.

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Extra resources for Activity-Based Proteomics: Methods and Protocols

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3. Add 50 μL STSB and centrifuge at 400–800 × g for 1 min (see Note 59). 4. Add 50 μL STSA and centrifuge at 400–800 × g for 1 min. 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).

Wear a lab coat, gloves and protective glasses at all times. 16. 5. In the absence of a substrate, trypsin tends to cleave itself (autolysis). To prevent this, trypsin is stored in solutions at low pH and at low temperatures. 5 μg/μL solution at −20 °C until needed. 5. The reaction buffer must be able to buffer out 50 mM acetic acid or HCl. 17. Calculate beforehand how much of the working solution you will need and prepare only this amount plus a little pipetting surplus. For example, if you have 20 in-gel digests and you need 20 μL of the working solution per digest, prepare (20 + 2) × 20 μL = 440 μL of the working solution.

Excise the marked bands of your gel with a new disposable steel blade (see Notes 37 and 39) and transfer the gel piece to an autoclave bag. 5 mL Eppendorf tubes. 5 In-gel Digestion 1. Wash the gel pieces twice with 500 μL water for 15 min while vigorously shaking (see Note 40). Briefly centrifuge (10 s, 16,000 × g) and discard the supernatant. 2. Wash the gel pieces twice for 5–10 min with 500 μL 100 mM ammonium bicarbonate (see Note 41) while vigorously shaking. Briefly centrifuge (10 s, 16,000 × g) and discard the supernatant.

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