Radiolabeled Ligand Binding Assay on 32D Cells

�(¹²⁵I erythropoietin or leptin)

Setup

� Make Binding Buffer (BB).� Use RPMI plus 0.1% BSA.

(The composition of BB can vary.� Other BBs are� unsupplemented RPMI, PBS, or DMEM plus 0.1% to 0.5% BSA.� Sterile filter. Store at 4�C.� Keep Sterile)

� Dilute radioactive ligand to 10⁴ cpm/ml in BB.

��������������� (Fresh leptin arrives at ~2x10⁴ cpm/ml, so add 5ml/10 ml BB)

��������������� (Fresh epo arrives at ~4x10⁴ cpm/ml, so add 2.5ml/10 ml BB)

Binding

� Into a 15 ml conical tube, transfer ~15 ml of confluent (1x10⁶ cells/ml) for 1-2 x 10⁷ total cells

� Spin at 2000 rpm for 5 minutes

� Aspirate media

� Resuspend in 1 ml of dilute ligand solution

� Transfer cells to 12 x 75 mm glass tubes

� Allow to sit for 45 minutes

� Spin at 2000 rpm for 5 minutes

� Aspirate media into a radioactive waste vessel

� Wash cells with 1 ml of PBS.� Spin down.

� Aspirate media into a radioactive waste vessel

� Wash cells with 1 ml of PBS. Spin down.

Counting

� Place tubes in gamma counter rack

� Add one tube containing 1 ml of dilute ligand solution for total counts

� Always include a negative 32D alone and positive (clone known to bind your ligand) in the assay.

� Count on program #39

Last updated 11/3/98.� AB