Blocking Protocol

1) Transfect 293 cells or Cos cells or any easily transfectable cell line with SR-BI.

2) Next day, add DMEM with 0.2% BSA to the media plus 1:500 dilution (or 1:1000) dilution of the SR-BI blocking ab. Incubate for 30 minutes to 1 hour at 37 deg C.

3) Add 1 to 10ug/ml of radiolabeled or fluorescent HDL (labeled either on the lipid or protein) to cells for 1to 2hours (in the presence of the blocking antibody). For control cells, do not add blocking aantibody.

4) Wash cells 3 to 4 times with ice cold PBS.

5) Measure HDL uptake by appropriate method (depending on label on HDL).

Note: As a positive control, you can add an excess (100-fold more) of unlabeled HDL to cells together with the label. This should block the uptake of labeled HDL by 80% or more. This positive control should tell you that your cells are expressing functional SR-BI. Also, cells not receiving either unlabeled HDL or no blocking ab should tell you that your cells are expressing functional SR-BI.