FAQ

The lipid components of the lipoproteins are susceptible to oxidation. The buffer we use acts as a preservative that deters oxidation from metal ions.

Once oxidation has begun, there are many fast acting, short lived, reactive products generated that interact with the lipid and /or protein portion of the particle. Changes may occur over days to weeks regardless of the buffer used for storage.

The purified lipoproteins may begin to aggregate after storage. Rough handling (such as vortexing or rapid pipetting) will accelerate this process. After months of storage, precipitates may form and the concentration of lipoproteins in solution will decrease. Fluorescently labeled lipoproteins will lose fluorescence intensity after a several months of storage. They are also prone to aggregation (see above).

The protein concentration is determined by the Pierce 660 nm Protein Assay using bovine albumin / gamma globulin for the standard curve.

The protein concentration determined by another assay or using another standard curve may give different results.

The products are provided in a buffer containing EDTA as a preservative and sealed under nitrogen gas when packaged.

• To prolong the shelf life of lipoproteins use aseptic technique whenever removing an aliquot.
• Handle the liquid gently (no vortexing) to prevent aggregation.
• Do not freeze the products to avoid aggregation.
• Do not allow fluorescent labeled lipoproteins to be exposure to light for prolonged time periods.

You can detect the fluorescence using filter settings for the following:

Specific configurations are available from the equipment manufacturers:

DiO is excited at 488 nm and detected with FITC emission filters (eg.533nm/30 or 525nm).

DiI is excited at 488 nm and detected with PE emission filters (eg.585nm/42).

We do not routinely assay for the cholesterol content of our Lipoprotein Depleted Fetal Bovine Serum. Our routine release testing includes the comparison of the starting serum with the depleted serum by agarose gel electrophoresis. We stain the gel with Sudan Black (a lipid stain) and confirm that the lipoproteins are depleted.

To check our depletion process, we have compared one preparation of the starting serum and depleted serum to verify that the cholesterol is indeed reduced. Using the HDL and LDL/VLDL Cholesterol Assay Kit from abcam (ab65390),we found a forty fold reduction.

Here are the results for that assay: