Example fractionation dating

To learn more or modify/prevent the use of cookies, see our Cookie Policy and Privacy Policy.We use/store this info to ensure you have proper access and that your account is secure.The C2C12 mouse skeletal myoblast cell line was obtained from the American Type Culture Collection (CRL-1772).C2C12 myoblasts were maintained in DMEM (Sigma Aldrich, Poole, UK) supplemented with 1% L-glutamine (Lonza, Cologne, Germany), 10% FBS (Biosera, Sussex, UK) and 1% penicillin and streptomycin (Sigma) under an atmosphere of 5% CO in humidified air at 37°C.This method permits samples to be frozen at −80°C for future analysis and/or additional processing at a later date.Isolation of nuclear, cytosolic and mitochondrial fractions of reasonable purity from mammalian tissues and cells has generated great interest as it has the advantage of allowing different cellular proteins and organelles to be studied and characterised.

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.

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