cell fractionation and low protein concentration in cytosol - (Nov/20/2008 )
I'm fractionating Hepg2 cells. Concen: Total cell lysate = 0.96 mg/ml, cytosol+membranes (supt of TCL spin 1000gx5min)=0.211 mg/ml, and crude nuclear fraction (pellet)=0.970. How could that be possible? Cells were lysis (TKMS) wasn't bad considering high conc in TCL but why CNF containing high conc of proteins? Aslo, CNF is very viscious when loading into the gel, how to make it less viscious? TKMS has Mg which was said to be a factor in stabilizing DNA.
-fred1972-
QUOTE (fred1972 @ Nov 20 2008, 01:37 PM)
I'm fractionating Hepg2 cells. Concen: Total cell lysate = 0.96 mg/ml, cytosol+membranes (supt of TCL spin 1000gx5min)=0.211 mg/ml, and crude nuclear fraction (pellet)=0.970. How could that be possible? Cells were lysis (TKMS) wasn't bad considering high conc in TCL but why CNF containing high conc of proteins? Aslo, CNF is very viscious when loading into the gel, how to make it less viscious? TKMS has Mg which was said to be a factor in stabilizing DNA.
the concentrations are very low for all your fractions. the least I get is 2 ug/ul ( 2 mg/ml)
-Curtis-