By Adam P. Arkin
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5%. 8. Heat for 8 min at 90◦ C in a heating block. CAUTION: Loosen caps before heating. 9. Allow samples to cool to room temperature. Proceed directly to Basic Protocol 2 or freeze samples at −20◦ C. ) injection using a CC-ABPP probe bearing a bio-orthogonal alkyne. Following homogenization (Support Protocol 1), a biotin-azide tag is then appended using CC. Note that (1) a no-probe control sample should always be prepared for comparison to the experimental sample; (2) if labeling enzymes with an azide probe and conjugating to a biotin-alkyne tag, the same protocol may be followed; (3) if labeling cell homogenates using CC probes, label the experimental sample aliquotted in ALTERNATE PROTOCOL Click Chemistry Activity-Based Protein ProÞling (CC-ABPP) 31 Current Protocols in Chemical Biology Volume 1 step 5 (below) with 5 to 20 μM probe-alkyne for 1 hr (using the same volume of DMSO for the control), and proceed directly to step 6.
10. 5% SDS in Ca- and Mg-free D-PBS to the protein pellet and sonicate three times, each time for 5 sec using the technique described in step 9. At this point, one may store samples at −20◦ C or proceed to next step of the protocol. 11. Heat sample for 5 min at 60◦ C in a water bath or heating block and repeat sonication two to three times, each time for 5 sec, to dissolve pellet. Centrifuge for 4 min at 6500 Click Chemistry Activity-Based Protein ProÞling (CC-ABPP) 33 Current Protocols in Chemical Biology Volume 1 × g, room temperature, to pellet any unsolubilized protein, and transfer supernatant to clean 15-ml conical tube.
26:2034-2035. Y. 2001. Genetically encoded reporters of protein kinase A activity reveal impact of substrate tethering. Proc. Natl. Acad. Sci. A. 98:14997-15002. Y. 2005. Insulin disrupts betaadrenergic signalling to protein kinase A in adipocytes. Nature 437:569-573. Kinase Signaling Visualized by Targeted Reporters 28 Volume 1 Current Protocols in Chemical Biology Activity-Based Protein ProÞling (ABPP) and Click Chemistry (CC)–ABPP by MudPIT Mass Spectrometry Anna E. Speers1 and Benjamin F. Cravatt1 1 The Skaggs Institute for Chemical Biology and Department of Physiological Chemistry, The Scripps Research Institute, La Jolla, California ABSTRACT Activity-based protein proÞling (ABPP) is a chemical proteomic method for functional interrogation of enzymes within complex proteomes.
Current Protocols in Chemical Biology 2009 (Volume 1) by Adam P. Arkin