Oxysterol binding assay
From LipidomicsWiki
[3H]OXYSTEROL BINDING ASSAY
This assay can be used for either cell extracts or purified proteins
Preparation of cell extracts
Cell are rinsed once with cold PBS, scraped in PBS (1 ml for a 60 mm dish) and collected by centrifugation.
The cell pellet is homogenized in the following buffer (use 100 µl for each 60 mm dish ) by 25 passages through a 25 Gauge needle. From a transfection of two 60 mm dishes of HEK293 cells using Lipofectamine 2000 we get approximately 200 µl cytosol, and from this we do one set of assays (4-5 conc hot + 4-5 conc hot/cold mix). Take apart 10 µl of the cytosol to check protein expression level by Western blotting. In assays with purified protein, we have used a final protein conc 1 µM (in some cases when little protein available, also 0.1 µM).
Binding buffer
10 mM HEPES (pH 7.4)
50 mM KCl
5 mM DTT
Protease inhibitor cocktail (Roche 11 836 170 001)
The resulting homogenate is centrifuged for 45 min at 100,000xg. Cytosol (supernatant) is collected and used to measure [3H]oxysterol binding activity.When using purified proteins, we have used the same binding buffer but without the protease inhibitors.
Preparation of [3H]oxysterol solutions
The stock [3H]oxysterol solutions are prepared as two mixtures; one contains the labelled sterol (hot mix), the other contains the labelled sterol plus a 40x excess of unlabelled 25-OH (hot/cold mix). The standard concentrations for a binding curve are (2.5), 5, 10, 20 and 40 (sometimes 80) nM [3H]oxysterol.
Stock solutions are prepared that contain 50x the above series of [3H]oxysterol concentrations (hot mix), and the same series of 50x stocks but with 40-fold excess of the corresponding unlabeled oxysterol. Thus the same volume of [3H]oxysterol is added to each assay (1.5 µl). Both hot and hot/cold mixes are prepared in ethanol
Binding assay
The assay is in a final volume of 75 µl with a final protein concentration of 0.3-0.6 mg/ml. Use 1.5 ml eppendorf tubes.
The binding assay is performed in binding buffer (see above) except that the KCl concentration is adjusted to 200 mM and polyvinyl alcohol (PVA) is added to a final concentration of 2% (w/v). Use a 20% PVA stock and 2M KCl stock to adjust to these concentrations. The volume of each assay can be adjusted to 75 µl by the addition of binding buffer.
All assays (hot and hot/cold mix) are performed in duplicate.
Assay is initiated by the addition of [3H]oxysterol (1.5 µl/tube) and incubated in a roller overnight (16h) at 4oC.
The next morning, transfer 65 µl of the assay to a new eppendorf tube and add 30 µl of the dextran/charcoal mixture (see below). Vortex at 5 min intervals for 30 min (at room temperature). Centrifuge for 10 min at 3500 x g in the cold room. Transfer 40 µl to a scintillation vial, add scintillation liquid and count.
Your will get two sets of data points for the oxysterol concentration curve: the hot mix gives total binding, while the hot/cold mix represents nonspecific binding not competed by excess cold oxysterol. Hot minus hot/cold curves give the specific [3H]oxysterol binding activity.
Some practical hints
In doing the assays, we always use a 60 µl total vol. of purified protein or cytosol, plus Binding buffer per assay
-6 µl of 2 M KCl/assay
-7.5 µl of 20% polyvinylalcohol/assay
-1.5 µl of [3H]oxysterol dilution
We have often used 5, 10, 20, 40, and 80 nM final conc of [3H]oxysterol (plus corresponding mixtures with a 40-fold excess of unlabeled oxysterol). With the labeled oxysterol preparations we have, we have had 150,000 – 200,000 dpm per assay at the 80 nM conc.
Our charcoal is Norit A Supra Eur cat no 94014-6 (Norit, the Netherlands) and the dextran Dextran T500, cat no 17-0320-01 (Pharmacia Fine Chemicals, Sweden).
The charcoal-dextran is prepared exactly as described in [Taylor, F. R. and Kandutsch, A. A. (1985) Use of oxygenated sterols to probe the regulation of 3-hydroxy-3-methylglutaryl-CoA reductase and sterologenesis. Methods Enzymol. 110, 9-19], and can be used for 1-2 months after preparation (storage at +4 C).
