Radiolabeled Ligand Binding Assay on 32D Cells
(125I erythropoietin or leptin)
(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)
·
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.
·
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