LDL isolation - (Aug/04/2004 )
I plan to isolate LDL from whole blood. Is there any guy can send me the protocol about isolation? I appreciate your help.
First you need to separate cells from plasma, 1500g 10minutes.
To remove the VLDL from the plasma, adjust plasma density to 1,019g/ml by adding KBr, the amount to be added is calculated by the following equation: g(KBr) = Vol (plasma) in ml *(1,019g/ml - density of plasma)/(1- (0,312*1,019)). The plasma is now going to be centrifuged (we use 40000 rpm in a Beckman L8-55 and a Ti60 rotor for 20h). The VLDL is now in the top fraction and can be discarded. The remaining plasma has the density adjusted to 1,063 by addition af KBr using the equation: g(KBr) = Vol (plasma) in ml *(1,063g/ml - density of VLDL deficient plasma)/(1- (0,312*1,063)) and centrifuged as above, now LDL is in the top fraction.
For measuring the density you need a picnometer - a glass with a fixed and very precise volume.