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

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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 suggestions in activity-based proteomics delivering a concise review of activity-based protein profiling. Written within the hugely winning Methods in Molecular Biology series structure, chapters contain introductions to their respective themes, lists of the required fabrics and reagents, step by step, quite simply reproducible laboratory protocols, and tips about troubleshooting and warding off recognized pitfalls.

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

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E64-d (10 mM): E64-d is a membrane permeable inhibitor of papain-like cysteine proteases. 43 g/mol) to obtain a 10 mM solution. Make aliquots and store them at −20 °C for up to half a year. 6. Triton X-100 is a non-denaturizing detergent that helps keep proteins in solution. 7. 7× EDTA-free Protease inhibitor cocktail (PIC III) (Roche Diagnostics). The protease inhibitor cocktail is provided in tablet form. Dissolve the tablet in ddH2O following the manufacturer´s instructions. Aliquots of 20 μL are stored at −20 °C.

Always work in a chemical hood especially when boiling samples containing β-mercaptoethanol. 10. Only tiny amounts of bromophenol blue are required. It is added as a pH indicator. 6, bromophenol blue containing solutions will be violet-blue. If the pH drops to a lower level, the color of the indicator will change to yellow [23]. 11. Cy2: λex = 489 nm, λem = 506 nm; Cy3: λex = 550 nm, λem = 570 nm; Cy5: λex = 649 nm, λem = 670 nm. Lasers: Blue 476 nm; Green 532 nm; Red 635 nm. 12. Steel blades are extremely sharp.

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. 3. Add 200 μL 10 mM TCEP or an amount sufficient to completely cover the gel slices. Incubate at 62 °C for 30 min (see Note 42).

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