also, my LDL solution becomes very much turbid while oxidation with CuSO4 for 24 hrs at 37 degree. Is the protein getting denatured or what ? Do, I have to spin it after oxidation to put down the turbid matter ?

Your LDL should lose its orange colour, but not go turbid. What do you dialyse your LDL into and how many times do you change your dialysis buffer? What concentration of CuSO4 do you use?

The same questions: what concentrations of LDL and Copper did you use? What is your initial Optical Density?

ok it's like this. I make the density of plasma with KBr and do the ultra centrifugation. Then I remove the LDL-HDL layer and again repeat ultra centrifugation, and then I get a good layer of LDL. (from 300ml of plasma I get around 10-12ml of LDL). Then I do the dialysis overnight at 4 degree in buffer which is 0.9% PBS solution in water. I go the dialysis in 2 L of buffer and I don't change the buffer. then after the dialysis, I measure the protein concentration with BCA (which is around 2-3mg/ml). I adjust it to 400µg/ml and do the dialysis with CuSo4 final conc. of 5µM at 37 degree for 24 hrs with shaking, After this step, my LDL looks very turbid, hazy and also there are lot of fine strands which I can see in the LDL. It seems to me that the protein is getting aggregated. I think so. So, before conc. the protein I have to actually spin it at high speed so as to get all the haziness and clumpy thing down otherwise I am not able to concentrate it using the 50ml colum filter.

Do you really need a concentration of LDL about 0.8 micromolar? You final OD is expected to be about 8 which is much higher than spectrophotometer is able to measure if you use 1cm optical pathway. You really "do the dialysis" with copper sulphate? We usually introduced copper salt into spectrophotometric cuvette.

There is not enough information in your question. Did you want to remove EDTA and salts with dialysis? The volume of buffer should be large (2 L) in comparison with LDL and it has to be changed at least twice in this time interval. Usually after dialysis you have to concentrate LDL dispersion and measure its concentration. What is the concentration of CuSO4 and its ratio to LDL? If you want to study slow oxidation, that is less than 1 metal ion per LDL particle, incubation with copper should be longer 2-3 days. You can look my article in Chem. Phys. Lipids 2004 (129) 75

If you add too much of copper ions, the rapid oxidation could denaturate protein, because it is known that much of radical damage is stored in protein domain.

Moreover, at high LDL, concentration of the dissolved oxygen may become the limiting factor for LDL oxidation.

I am sorry for my mistake "I adjust it to 400µg/ml and do the oxidation with CuSo4 final conc. of 5µM at 37 degree for 24 hrs with shaking,

My main problem is during oxidation the solution of LDL becomes hazy turbid thread like. CuSO4 final conc. is 5µM. LDL i get after dialysis is 2-3mg/ml, and I dilute it to 400µg/ml and then do the oxidation with CuSO4 (5µM final conc.), 37 degree shaking.

Try 0.1 micromolar LDL at the same CuSO4. In any case, what optical density you had before adding copper? What is the need for 24 hours one-point measurement? According to my experience, the main events occur during first 2-3 hours.

I think you need some more dialysis buffer changes to make sure all the KBr is removed, before exposing the LDL to CuSO4, and that the shaking may be a problem. This may be what is making the LDL aggregate, so try gently mixing the LDL by inverting the tube, and let it sit for the rest of the 37 C incubation.

I guess that 400 µg/ml concentration of LDL is fine but u may reduce it if possible (try 50-100 µg/ml). What is the shaking rpm? If the speed is high, automatically LDL gets precipitated.

One thing might be also possible that oxidation is too high and so protein gets precipitated but with this concentration of CuSO4 I wouldn't assume that.

If you check OD after 3 h oxidation, it should increase from 0.8 to 0.9 at least.

If you start the isolation from EDTA-plasma, the dyalisis for sure is insufficient. Then no copper would be available to oxidize your LDL. Then i would for sure discard the agitation during incubation. Then, check again the concentration of LDL. Why is the need of the 24 hs incubation?(total LDLoxidation with that conc. of Cu2+ is achieved around 3hs). It also could happen that your LDL is already oxidized during the process. Take special care to use N2 to remove air when you are storing the tubes meanwhile you are doing other things. Personally i also, use N2 before closing the dyalisis clips.

Adding to Tomás Meroño answer: you can see several contributions in my profile. One of these is: Nitric oxide inhibits prooxidant actions of uric acid during copper-mediated LDL oxidation.

Silvia M Sanguinetti, Carlos Batthyány, Andrés Trostchansky, Horacio Botti, Graciela I López, Regina L W Wikinski, Homero Rubbo, Laura E Schreier

See also: Influence of the medium on the assessment of LDL resistance to oxidation: lag time in phosphate buffered saline is longer than in sodium chloride solution.

L Schreier, F Pagliero, S Sanguinetti, R Wikinski

Atherosclerosis 03/1997; 129(1):127-8. · 3.79 Impact Factor

Both articles and others are included in my profile and Tomás Meroño'profile

how many changes on you dialysate? likely you have some antioxidant remaining in the sample. Change 3 x and make sure you have at least 20:1 dialysate:sample. Make sure there are no antioxidants in the dialysate buffer (e.g. EDTA). What is the copper concentration? Jack it up, if you cannot burn the lipoproteins at say 40uM perhaps your starting material is already fairly oxidized. What is you initial OD at 234? Make sure your spec is working properly.

what would be the best buffer to do the dialysis ? I use 0.9% PBS solution in water.

Make sure you also don't have BHT in your original LDL isolation protocol, as BHT will inhibit oxidation. For the first time last week I came across an LDL protocol that had BHT added at some stage, and that would obviously prevent your oxidation.

I dialyze the LDL against PBS without EDTA for 24h changing the PBS 3 times. For 1mL of LDL include more 3ML of PBS without EDTA and incubate with 40 microliter of 1mM CuSO4 at 37 ºC for 24h. It is important no close the recipient (it's important the contact with the air) and keep under gentle agitation.

Good luck

After ultracentrifugation I usually isolate LDL from EDTA, through columns of Bio-Rad with Bio-Gel P6, and immediately add CuSO4 to LDL for oxidation in 37C. Without dialysis, my LDL oxidation goes perfectly fine. For any details please do not hesitate to contact me in P.M.

Hi Kali.. Can I use this protocol for the oxidation of specific (purified) cholesteryl esters such as cholesteryl oleate?

Hi Neha. I have never tried that, so I am sorry but I cannot advise you on such an option.

Hi Kali,

How can I check whether my LDL has been oxidized or not after after 24 h,

LDL incubation using CuSO4 for 2h ?

It is difficult to find out the LDL oxidation process at 234 nm. You will get nice conformation by Thiobarbituric Acid-Reactive Substances (TBARS) assay.

• Preparation of Oxidized LDL :-
• Dissolve LDL (5 mg) in PBS (1 mL). Add PBS (1 mL) containing CuSO4 (10 μM). Transfer to 3.5 cm sterile plastic culture dish and incubate for up to 48 h at 37°C. Dialyze against 12 L PBS for 48 h at 4°C (two changes of 6 L each). Determine protein content by the Lowry method.
• Measurement of LDL Thiobarbituric Acid-Reactive Substances (TBARS) in Oxidized LDL . Add LDL (50 μg) to NaCI (150 mM, 1.5 mL) in a 13×10 cm culture tube. Add TCA (20% w/v, 0.5 mL) and TBA reagent (0.5 mL). Boil at 95°C for 1 h. Cool with tap water. Add butan-1-ol (2.0 mL) and vortex mix vigorously. Centrifuge at 4,000 g for 15 min. Remove top layer and measure fluorescence (excitation at 515 nm, emission at 550 nm).
• For more detail protocol is written in our recent published journal. See the link below.

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