By Adam P. Arkin
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Extra info for Current Protocols in Chemical Biology 2009 (Volume 1)
Store at room temperature for days or 4◦ C for weeks. Prepare a stock of probe-alkyne in ethanol as follows. , if desired dosage is 10 mg/kg, then concentration would be 20 mg/ml; typical dosage is 10 to 50 mg/kg). Assuming a maximum mouse weight of 30 g, prepare 15 μl of ethanol solution per mouse. Prepare a probe-alkyne solution in 18:1:1 saline:castor oil:ethanol as follows. , 1 mg/ml from a 20 mg/ml ethanol stock) in 18:1:1 saline:castor oil:ethanol. Ten μl of this solution should be injected per gram of mouse.
Dry sample in a SpeedVac evaporator. 16. Resuspend peptides in 100 μl of 5% formic acid and vortex 5 min. Proceed directly to MS analysis or store samples at −80◦ C (stable for up to several months). 17. Analyze by MudPIT. For additional details on analysis of labeled peptides, see Link et al. (2003) and Weerapana et al. (2007). Click Chemistry Activity-Based Protein ProÞling (CC-ABPP) 37 Current Protocols in Chemical Biology Volume 1 REAGENTS AND SOLUTIONS Use Milli-Q-puriÞed water or equivalent for all aqueous solutions used in this unit; for suppliers, see SUPPLIERS APPENDIX.
After 1 hr, sacriÞce mice according to approved animal protocol and harvest tissue. 4b. Snap-freeze tissue on dry ice. Process tissue (prepare homogenate; see Support Protocol 1) for biotin-azide conjugation, or store samples at −80◦ C (stable for months). Perform biotin-azide conjugation via click chemistry 5. 5-ml portions of 2 mg/ml cell/tissue homogenate (prepared in Ca- and Mg-free D-PBS, see Support Protocol 1) into microcentrifuge tubes. The CC reaction works best in Ca- and Mg-free D-PBS.