Fig. 3. SDS gel electrophoresis of myotube proteins. (A) A 3-7.5% gradient protein gel for the targeted clone (lanes 1-3, separate protein samples) and wild-type clone (lane 4) that has been silver stained. For both wild-type and the targeted (c20) clones, two bands for titin can be seen. In the targeted clone, the lower band is approximately 0.2 MDa smaller than the corresponding band in the wild-type clone. This reduction in size is consistent with the targeted deletion of the C-terminal end. Protein samples were not equally loaded for this gel resulting in some intensity variation for the bands for c20 samples. Approximate molecular masses are shown on the right, in MDa. (B) A western blot of 2-7.5% gradient protein gel for heart muscle (lane 1), wild-type 5-day myotubes (lane 2), and targeted (c20) 5-day myotubes (lane 3) using the Z1/Z2 antibody, against the N-terminal domain of titin. The isoform expressed in heart muscle is approximately 2.8-2.9 MDa, and can be seen as a single band. In the myotubes, there are two immunopositive bands for titin in both the wild-type and the targeted (c20) clones. The difference in sizes for the two bands in the wild-type and targeted clones cannot be resolved on this blot. (C) A western blot of a 2-7.5% gradient protein gel for wild-type (lanes 1 and 3; two separate protein samples), and clone 20 (lanes 2 and 4; two separate protein samples). Two bands for titin kinase can be seen in both the wild-type samples, however, only one band can be seen in both c20 samples when probed with the anti-kinase antibody. This gel was run for longer than the gel in B, hence the larger separation between the two bands present in the wild-type clone. This suggests that the lower band in c20 is not immunoreactive to the kinase antibody.