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