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