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 ideas in activity-based proteomics offering a concise evaluation of activity-based protein profiling. Written within the hugely profitable Methods in Molecular Biology series structure, chapters comprise introductions to their respective issues, lists of the mandatory fabrics and reagents, step by step, effortlessly reproducible laboratory protocols, and pointers on troubleshooting and keeping off identified pitfalls.
Authoritative and state-of-the-art, Activity-Based Proteomics: equipment and Protocols goals to make sure winning leads to the additional examine of this important field.
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Extra resources for Activity-Based Proteomics: Methods and Protocols
TCEP will reduce disulfide-bonds (R1-S-S-R2 → R1-SH + HS-R2) which is essential for the subsequent cysteine alkylation . You can also use other reducing agents like DTT and BME. 43. The IAA treatment is necessary to irreversibly alkylate the thiol-group of cysteins (R-SH + I-CH2-C(O)-NH2 → R-SCH2-C(O)-NH2 + HI) which are prone to oxidation . This chemical reaction leads to a uniform a 57 Da carbamidomethyl modification of all thiol groups (-SH) which facilitates the automated detection of cysteine-containing peptides by MS.
Use this working stock immediately. Do not store this solution for longer periods (see Note 17). 9. 33 mM FA. Mix 50 μL 100 % FA (HCOOH) in 950 μL MS water. 10. 27 mM FA. Mix 1 μL FA with 999 μL MS water. 6 Micropurification of Samples on C18 StageTips 1. 200 μL C18-StageTips: self-made or commercial (Thermo). 2. Methanol (MeOH), 100 %. 3. Acetonitrile (ACN), 100 %. 5. 4. Formic acid (FA), 100 %. 5. No. 5010 21514). 6. 5 % (v/v) FA in H2O. Mix 10 μL FA and 1980 μL MS water in a 2 mL Eppendorf tube.
Make 1 mL aliquots and store these at −20 °C. 7. Gel loading tips. 8. KIMTECH paper towels (fluff-free). 9. Laser scanner or imaging camera with appropriate filter system to detect the fluorescent dye of the reporter. We use a Typhoon FLA 9000 Scanner from GE Healthcare. This scanner comes equipped with the filter settings and lasers for Cy2, Cy3, or Cy5 or similar dyes (see Note 11). Imaging systems are usually less sensitive and they do not allow to image large gels. 10. Transparent autoclave bag.
Activity-Based Proteomics: Methods and Protocols by Herman S. Overkleeft, Bogdan I. Florea