Example fractionation dating
This enabled a comparison of the efficacy of the commercial kit with the method described here when both methods were used to fractionate an identical starting sample (myoblast cells from a T75 cm cells).
To induce myogenic differentiation, the growth medium was changed to differentiation medium (DMEM supplemented with 2% horse serum (Sigma) and 1% antibiotics) after myoblasts had reached ≈ 90% confluence in a T75 cm flask.
Myoblast cells were either harvested at 90% confluence or allowed to mature to myotubes for 7 days and then harvested (see below).
All samples were resuspended in 300-500 μl of STM buffer comprising 250 m M sucrose, 50 m M Tris–HCl p H 7.4, 5 m M Mg Cl, protease and phosphatase inhibitor cocktails (all chemicals were from Sigma-Aldrich, Poole, UK unless stated otherwise) and homogenized for 1 minute on ice using a tight-fitting Teflon pestle attached to a Potter S homogeniser (Sartorius Stedium, Goettingen, Germany) set to 600–1,000 rpm.
The homogenate was then inspected, if intact tissue was still evident the homogenisation was repeated.