TITLE: Ultracentrifugal separation of VLDL, LDL and HDL
AUTHORS: Daniel Teupser, Jan Breslow

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">This protocol is used to isolate the various lipid fractions from blood plasma using ultracentrifugation. The actual measured concentrations are performed separately once the isolations are complete.</div><div class = "text-block"><span style = "font-style:italic;font-weight:bold;">NOTE: This protocol is not applicable for ApoE knockout mice.</span></div><div class = "text-block"><span style = "font-weight:bold;">Diabetic Complications:</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bakqr6.jpg" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bakqse.jpg" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bakqsn.jpg" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bakqsw.jpg" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bakqs6.jpg" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bakqte.jpg" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bakqtn.jpg" /></div></div>

[STEPS]
?. Add 60 ul of plasma to Beckman ultracentrifugation tube (7 x 20 mm; thick walled; polyallomer; cat. # 343621).
?. Layer 60 ul of PBS on top of the plasma and place tubes in a TLA100 rotor.
?. Spin for 3 hours Beckman Optima TL tabletop ultracentrifuge at 70,000 rpm, 4°C.
?. Using a 100 μl Hamilton syringe, carefully remove the bottom 60 μl and transfer to a new Beckman tube labeled with the sample number and A. Between samples rinse the Hamilton syringe with distilled water.
?. Using a rinsed Hamilton syringe transfer the rest of the sample (upper portion) into a second tube labeled with the sample number and B.
?. Add 60 μl KBr solution (density = 1.12 g/ml) to tube A to make a final density of 1.063 g/ml) and mix 5 to 6 times up and down with the same pipette tip.
?. Layer 60 μl of PBS on top of the sample in tube B.
?. Spin both A and B for 18 h overnight in the ultracentrifuge at 70,000 rpm at 4C as above.
?. Using a rinsed 100 μl Hamilton syringe remove the bottom 60 μl from tube A and transfer to an Eppendorf tube labeled HDL. Using a rinsed Hamilton syringe transfer the remaining 60 μl (upper portion) to an Eppendorf tube labeled LDL.
?. Using a rinsed Hamilton syringe remove the bottom 60 μl from tube B and transfer to the same Eppendorf tube labeled LDL in step 9 above (To recover any LDL contaminating the VLDL preparation after the first ultracentrifugation spin).
?. Using a rinsed Hamilton syringe transfer the remaining 60 μl from tube B to an Eppendorf tube labeled VLDL.
?. Measure cholesterol, triglycerides or phospholipids concentrations in the lipoprotein fractions using their respective protocols.The densities of the fractions are as follows:VLDL LDL, IDL 1.006 – 1.063 g/ml HDL > 1.063 g/ml