Do you have ultracentrifugation? You must keep the good gradient ( you must use KBR to this)

I agree with Marta, but if you use KBr, you will need to dialize the fractions afterwards, as it can be toxic for the cells. Isolating VLDL is quite easy but I have no experience in isolating HDL fractions for subsequent use. This can be rather tricky as it is difficult to obtain fractions concentrated enough.

That's right, that my procedure requires dialysis. But if it is a double dialysis (once in Tris-HCl + 0.9% NaCl, only the second time in Tris-HCl) fraction is pure.

Thanks for the replies!

I think I will order them.., I think it's rather complicated to get all the fractions out otherwise.. They need to have a high purity, and indeed, i don't want any toxic subtances..

However, when I order them, they are in a solution that contains EDTA. I would like to oxidize a part of the lipoproteins (comparing oxidized vs non oxidized lipoproteins in cell cultures) with 5 micromolar CuSO4.., that may be a hard job when EDTA is present I guess :-)..? Or maybe I can add more CuSO4 to overcome this problem?

I use this protocol: 1. adjust plasma density with KBr to 1.063 g/cm3 and put in ultracentrifuge, after 24 hours you will get VLDL+LDL at the top, water in the middle and HDL at the bottom; the gel precipitation are proteins, throw that away; 2. take VLDL+LDL fraction and adjust density to 1.050; after 24 hours at ultracentrifuge you will get VLDL+LDL at the top and remanining water below; 3. adjust density to 1.020 and after centrifugation you will ger VLDL (together with some IDL) at the top and LDL at the bottom; 4. centrifugation of HDL fraction adjusted to 1.21 will enable to get rid of remaining proteins; after that adjust HDL solution to 1.125 and after 48 hours you should ge HDL2 at the top and HDL3 at the bottom; often these two fractions are not well separated

Dear Gianpaolo Tibolla. I too am facing problem with LDL isolation. I am getting orange yellow band at bottom most part of tube. I am adjusting density of plasma to 1.21 g/ml with solid KBr as some protocols suggest. Also we have ultracentrifuge with 1 ml sample volume capacity and 1,40,000 rpm limit and fixed angle rotor. Is is sufficient to get pure LDL fraction. Please suggest.

Gianpaolo Tibolla.Please how can adjusting plasma to separate HDL. I need simple method.I am student

Gianpaolo Tibolla: Can you provide me with a protocol? I need to isolate HDL and LDL from mouse plasma. We have a ultracentrifuge but no one with experience in this area.

You can make Ultracentrifugation in density gradient, (KBr in the bottom with a density of 1.25 g/ml, on which is superimposed a solution of d = 1, 210 g/ml and above it another d = 1, 000). After Ultracentrifugation, the areas of interest are located, and it can be removed by puncture and aspiring UC tube band carefully (the upper band first and so one). This technique requires that we have previously performed tests to establish the curve of densities we get with our gradient design. After isolated may be necessary dialyzing the fractions obtained against buffer need to use.

This technique requires a SW41 (or similar rotor), 40000 rpm for 22h and appropriate Ultraclear tubes for sample volumes up to 3 ml, or SW60 (or similar rotor) and 60000 rpm for 18h, for small volumes.

Hi Winde,

Before oxidase lipoproteins, you need dialise the samples with PBS without EDTA for 24h change the buffer 3 times in these period, if you do not dialise, the oxidation is very dificcult. Before use in cell culture sterilize the lipoprotein with 0.22um siyringue filter. I work with oxidised LDL in my master degree, if you have more help please contact me.

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A crude procedure that may be useful if you want to avoid ultracentrifugation. You may use a gradient gel suitable for HDL isolation (if available commercially). Add your sample with added lipid stain in a big well and let it run. HDL bands may appear as a smear on the bottom of the gel depending on the gradient you use), Cut the HDL bands (smears). Electro-elute them and then dialyze the sample. But you need to control the volumes to get a suitable concentration of samples. The band in the middle is LDL depending on the gradient of the gel (it will be a different gradient if you want to prepare HDL). The band on the very top is mainly VLDL if you use a fasting serum sample. Also, it depends on how much sample you need. This is suitable to obtain small samples, and I am not sure if it will be easy to separate the HDL fractions. It is fine to separte VLDL, LDL and HDL. My experience is with separating small dense LDL. Just thought that this may give you a lead in your search.

However. I think that the procedure described by Gianpaolo is the best method.

In my opinion , as have already mentioned some researchers , the best method of separation is the UC density gradient of 1.23 g / ml to 1.000 g / ml. It depends on the rotor (preferably swingarm for better resolution ) , the starting volume may be 1 or 3 ml adjusted with KBr to a density 1,230 and with a soft prestaining with CBB to locate the bands . The separation is usually excellent (22h , 40000 rpm for SW41 , starting with 3 ml ) ,however we're having trouble isolating some bands, especially if they are close together , so that often double bands of LDL are usually collected together. You must then proceed to dialysis without EDTA..

One problem with this method is that if we want to work with HDL , we must be aware that during ultracentrifugation , some of its proteins ( HDL are released and appear in the LPDS ) are lost , so if you want to work with HDL highly conserved by the process , it is preferable FPLC separations : more tedious and less discriminative but better they retain the structure of HDL .

Dear Winde,

Alfa Aesar , now part of Thermofisher , offersa range of Lipoproteins manufactured inhouse.  I have attached our range, details on our website www.alfa.com

 I would like to isolate the HDL from plasma, there are several methods available, I want to know which one would be the best, It would be the first time to start in our lab, we have ultracentrifuge for separation, can anyone allow me to access for that protocol