Research Paper Volume 1, Issue 6 pp 557—572

Figure 6. The 130 kDa CKm species is 3-nitrotyrosine modified in an age-dependent fashion. (A) Blue Sepharose protein fractions #26-#32 (see Figure 5) that contained anti-CKm immunoreactive proteins with apparent masses of 88 (CKm 88) and 130 (CKm 130) kDa were pooled from young, middle-aged and aged mouse samples and individually loaded onto a 1 ml mono-Q-Sepharose column (Biorad Laboratories). After application, samples were washed with 10 ml of 25 mM Tris pH 8.0, developed with a shallow 20 ml linear NaCl gradient (0-250 mM NaCl in 25 mM Tris pH 8.0) followed by a steep 10 ml NaCl gradient (250 mM - 1M NaCl in 25 mM Tris pH 8.0). Flow rate equaled 1 ml/min throughout purification and fractions were collected at a rate of one fraction per minute. The above chromatograph was obtained by fractionation of the middle-aged protein sample, fractionation of the young and aged samples yielded similar chromatographs. Fractions 26-28 (indicated on the figure) contained CKm 130. These fractions were pooled and analyzed for 3-nitrotyrosine modification. (B) Pooled protein (0.5 μg) from young (Y), middle-aged (M), and aged (A) Q-Sepharose fractionations were resolved by SDS PAGE and transferred to a PVDF membrane. Blots probed with an anti-3NT antibody [top blot, panel (B)] reveal that CK 130 is 3-NT modified in an age dependent manner. The membrane was then stained with Coomassie Blue [bottom blot, panel (B)] to normalize protein loading. (C) Densitometry was used to compare the relative abundance of the 3-NT modified form of CK 130 between age groups.