Research Paper Volume 1, Issue 6 pp 557—572
Oxidative modification and aggregation of creatine kinase from aged mouse skeletal muscle
Figure 3. Chromatographic elution properties of 3-nitrotyrosine modified muscle creatine kinase are altered. (A) Immunoblots of Blue Sepharose fractions of CKm show that fractions 9-12 are tyrosine nitrated. CKm (1 μg, > 85 % pure) was resolved on a denaturing SDS polyacrylamide gel, transferred to a PVDF membrane, and analyzed by immunoblot using anti-3NT-antibody. (B) Immunoblots of Blue Sepharose fractions 3-7 shows that 3-NT CKm is not detected in these fractions. Blots probed with an anti-3-nitrotyrosine antibody (top panel) detected nitrated glycogen phosphorylase in middle aged and aged muscle. However, the purified CKm in this fraction is not nitrated. Blots were reprobed with an anti-CK antibody (3B, lower panel). (C) Fractions 9-11 were further fractionated by reverse phase chromatography. These fractions were spotted on to PVDF membranes and analyzed for nitrated CKm using anti-nitrotyrosine antibody, and carbonylated CKm using anti-DNPH antibody (D).