Extended Data Fig. 3: Purification and characterization of Sir2/Ago complexes.
a, MS analysis of wt GsSir2/Ago complex. The theoretical Mw of the GsSir2 and GsAgo proteins (without 1st Met) are 67929.51 Da and 53135.61 Da, respectively. b, CD spectra of wt GsSir2/Ago and mutants. Mutant spectra are similar to that of a natively folded protein. c, Size-exclusion chromatography of wt GsSir2/Ago, showing elution volume and comparing to mass standards. According to mass spectrometry of the purified GsSir2/Ago complex, the molar mass of the GsSir2/Ago heterodimer is 121 kDa. d, SDS–PAGE analysis of fractions containing the CcSir2/Ago complex eluted from Heparin column. Densitometric inspection shows that Sir2 and Ago proteins are in the ratio ~1:1. Single replicate. e, SDS–PAGE analysis of the CcSir2/Ago stock after dialysis against a storage buffer. Various amounts of the stock solution were loaded on the gel. Densitometric inspection shows that Sir2 and Ago proteins are in the ratio ~0.3:1. Single replicate. f, MS analysis of the CcSir2/Ago complex. The experimental masses (53174.71 Da and 67629.21 Da) are close to the theoretical molecular masses of the Ago protein (53173.91 Da) and the Sir2 protein with the truncated tag at the N terminus (67626.24 Da). g, SEC-MALS analysis of the CcSir2/Ago complex. The experimental mass of 124 kDa is close to the theoretical molecular mass of the CcSir2/Ago heterodimer (121 kDa). h, MS/MS calibration curve of NAD+ standard (marked in black) and the observed amount of NAD+ (marked in red) in the CcSir2/Ago complex (20 pmol according to the Ago protein). The discrepancy between the expected amount of NAD+ (20 pmol, marked in green) and the actual amount (6.45 pmol, marked in red) was due to the decrease of the Sir2 protein in the CcSir2/Ago preparation (see e).
