By Adam P. Arkin
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2005. Signal propagation from membrane messengers to nuclear effectors revealed by reporters of phosphoinositide dynamics and Akt activity. Proc. Natl. Acad. Sci. A. 102:15081-15086. Y. 1999. Circular permutation and receptor insertion within green ßuorescent proteins. Proc. Natl. Acad. Sci. A. 96:11241-11246. Y. 2002. A monomeric red ßuorescent protein. Proc. Natl. Acad. Sci. A. 99:7877-7882. J. E. 2009. Genetically encoded FRET-based biosensors for multiparameter ßuorescence imaging. Curr. Opin.
It should be noted that protein spectral counts in the tryptic dataset (rather than the labeled-peptide dataset) are used to estimate active protein abundance, as labeled peptides typically give too few (<10) spectral counts for reliable estimates of active protein. , modiÞcation of a catalytic residue). , 2005). , 2008) cleavage of tags with labile linkers. , 2003). However, direct elution of biotinylated peptides can be implemented with a high degree of success. , 2007). , 2002; Speers and Cravatt, 2004) prior to performing the ABPP-MudPIT protocol.
13. Analyze by MudPIT. For additional details on analysis of tryptic peptides, see Link et al. (2003) and Weerapana et al. (2007). Elute labeled peptides for MS analysis (optional) Note that it is not necessary to complete steps 14 to 17 unless analysis of probe labeling sites is desired. 14. Wash beads in a Bio-Spin column Þve times, each time with 1 ml Ca- and Mg-free D-PBS, then Þve times, each time with 1 ml water. Transfer beads to a clean, lowadhesion tube in water, spin brießy at maximum speed in a microcentrifuge, and remove supernatant.