Myosin Light Chain Lab Report

The objective of the experiment was to differentiate mammalian C2C12 cells into muscle cells and analyze for presence of myosin light chain. Utilizing Western Blotting techniques and using specific antibodies helped to detect myosin light chain. The hypothesis was that proliferating myoblasts could be induced to initiate the differentiation process by depriving the cells of the necessary growth factors and that the differentiation is characterized by the expression of muscle-specific proteins. The results showed that that myosin light chain was not present. It was concluded that the cells were not differentiated enough to begin with, the amount of cells used was not sufficient and the differentiation media did not have a neutral pH at the time of harvest.
Introduction
The purpose of the experiment was to differentiate C2C12 cells into muscle cells and tests for presence of muscle protein specifics. The hypothesis was that proliferating C2C12 cells could be induced to differentiate by removal of growth factors and differentiation would be characterized by expression of the muscle specific protein myosin light chain. The rationale is that only differentiated muscle cells
This allows an equal concentration of protein from each sample to be load into the wells later on when the gel electrophoresis is run. The Coomassie dye is brownish/red in color but turns blue when mixed with proteins. The blue color can be measured in a spectrophotometer at 595nm. A standard curve was generated using known amounts of bovine serum albumin (BSA). Four microfuge tubes were set up and 200ul of Bradford reagent were added to each tube and 1ul of whole cell extract was added to each appropriate tube. Entire sample of four tubes were transferred to four wells on 96-well microtiter plate. The Absorption at 595nm was measured for all of the samples and then