Brugia Malayi - Clark Science Center - Smith College

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Creatine Kinase during Muscle Development Hailun Li Creatine kinase (CK) plays a major role in catalyzing the conversion of phosphocreatine and ADP into creatine and ATP, which provides nearly instantaneous ATP regeneration to many cell types with a high energy demand. Thus, it is not surprising that CK is found in mature muscle cells, but when does it appear in the development of muscle cells? This summer, I quantified CK in the three developmental stages of the C2C12 cell line, myoblast (M), early myotube (EM), and late myotube (LM). Several techniques were used: cell culture; cell lysis; sample protein estimation (Lowry assay); protein separation through SDSgel electrophoresis, along with a CK standard curve; immunoblotting to a PVDF membrane, which was probed using an anti-CK primary antibody at 4°C overnight; followed by quantitative gel densitometric scanning. As I presented to my peers in the lab, CK exists at relatively low levels in all three stages (Figure 1). While a CK band is clearly observed on the lane with 0.2 μg of the adult skeletal muscle cell extract sample, it was not found in gel lanes with 20 μg of the myoblast, early myotube, or late myotube cell samples. Increasing the loadings to 50 μg of cell extracts, a band could be seen at relatively low intensity for each cell sample. Preliminary quantitation (Figure 2) demonstrates that CK exists at the highest level in early myotubes and the lowest level in myoblasts, although the differences between the stages are small in general. Despite the high level of creatine kinase in mature muscle cells, its low level in the developmental stages indicates that its increase in amount may happen relatively late in cell maturation. However, this result and the differences between the stages in the quantification should not be considered significant until the experiment is repeated with further optimized conditions. Aside from this, I hope to identify the band on each lane of the cell samples, study more about creatine kinase with 2-D gel techniques, and continue this project into the coming academic year. (Supported by the Howard Hughes Medical Institute) Advisor: Stylianos P. Scordilis Figure 1. An immunoblot using anti-mouse skeletal muscle creatine kinase antibody. The first four lanes are the standard curve using skeletal muscle creatine kinase, followed by C2C12 cell extracts. 2012 21
Figure 2. The integrated pixel intensity from an immunoblot using anti-mouse skeletal muscle creatine kinase antibody for 10 µg skeletal muscle creatine kinase, and 50 µg of myoblast (M), early myotube (EM) and late myotube (LM) C2C12 cell extracts. 2012 22
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