Acyl-ACP Ketosynthase Loading Assay

KS loading assays were conducted in storage buffer (25 mM Tris, 500 mM NaCl 10 % (v/v) glycerol, pH 7.6). Typically, 20 p,M KS was incubated with 100 ttM acyl-ACP and the reaction was allowed to proceed at 25 °C. Aliquots were removed at various time points between 2-32 h, and subsequent ZipTip clean-up of the samples allowed MS analysis of each time point (see Sect. 2.2.15.1). 

Hydrolase assays were conducted in storage buffer (25 mM Tris, 500 mM NaCl, 10 % v/v glycerol, pH 7.4) in the presence of GroEL. Typically, PedC (5 p,M) was incubated with acyl-ACPs (20 xM) in a 30 ttL reaction for various periods of time ranging from 0.5-60 min at 25 °C. The reaction was quenched using 0.1 % TFA, followed by ZipTip desalting for MS analysis (see Sect 2.2.15.1). 

7 Acyltransferase Extender Unit Spec city Assays 

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