Hi, I recently have tried to oxidize commercial native human LDL(Kalen biomedical).

But after oxidation procedure with 10uM copper sulfate for 18hrs, relative electrophoresis motility(REM) and TBARS assay results were poor.

REM was 1.2~1.4 and TBARS assay results were ~2.5nmol/ug of protein.

Even oxidation with 20, 30uM copper sulfate for 18 hrs showed more poor REM and TBARS assay results.

I have tried to find problems in my procedure and now I'm trying dialysing native LDL to remove EDTA not using PD-10 desalting column.

However, it is not easy to find out what the problem is.

Is there any tips for sufficient oxidation of commercial native human LDL?

My goal of REM and TBARS assay results of my oxLDL is 1.7-2.0 and 18-21nmol/ug protein.

My oxidation protocol is as followed;

1. Remove EDTA in native human LDL(from Kalen biomedical) by PD-10 desalting column following manufacturer's manual.

2. Dilute desalted native LDL to concentration of 1mg/ml with sterilized PBS.

3. Add 10mM copper sulfate solution [copper sulfate(sigma, C8027) in D.W] to native LDL

in 50ml cornical tube. Concentration of copper sulfate in native LDL is 10uM.

4. Incubate native LDL in 37°C water bath for 18 hrs w/o shaking and terminate oxidation by adding EDTA for 0.34mM.

5. Dialysis oxidized LDL with semi-permeable membrane(MWCO 12-14kDa) in 0.34mM

EDTA/PBS at 4°C for 72hrs. Change dialysis buffer for 5 times.(4hrs + 8hrs + 12hrs + 24hrs +

24hrs).