Dear Hannah

I am not familiar with the stability of S1P but a convenient method to isolate HDL is the precipitation of apoB containing lipoproteins with phosphotungstic acid and magnesium chloride. In 15 min it´s done whereas for an ultracentrifugation you need the technical equipment and much more time.

Good luck!

Dear Michael,

thanks a lot for that hint! I have read about that, too, it sounds like a practical method. Can you recommend a protocoll by which to operate? And maybe even a recipe to mix the precipitation reagent? 

Have you experienced any inaccuracy in samples with altered triglycerides?

Thanks in advance.

Dear Hannah

we are working with a precipitation solution of Roche (ordering number 10543004122; we pay 16 € for 100 ml) and according to the flyer the concentration of MgCl is 20 mmol/L and of phosphotungstic acid is 0.44 mmol/L.

You mix one part serum or plasma (we work normally with EDTA, therefore I can´t promise that it will work with citrat, too) with 2.5 parts of the reagenz, mix the solution thoroughly by vortexing, incubate 10 min at room temperature and centrifuge for 5 min at 10.000 rpm in a table top centrifuge. If the triglycerides are really high the method isn´t working well, you still might have floating chylomicrons or VLDL.

Here is what we use -

4) Dextran sulfate-magnesium chloride solution (HDL Reagent): Use 0.1 mL per 1 mL of plasma or serum.

Prepare 2 solutions that are mixed in equal volumes to make the precipitating solution (HDL Reagent):

Solution 1: 0.3g Dextralip 50 (dextran sulfate) in 15ml ddH2O

Solution 2: 1M MgCl2٠6H2O (FW=203.31g/m, 1M=203.31g/L = 3.0497g/15ml ddH2O

We used both phosphotungstate- magnesium chloride and dextransulfate -magensium Chloride. Both work well. Another possibility which we used is an immunabsorption column with anti APO B antibodies linked to sepharose as it is used for LDL apheresis.