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 4C. 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