General Protocol for an 125I Erythropoietin or 125I Leptin Binding Assay with 293 Cell Line




1.        These cells grow in DMEM-HIGH supplemented with 10% Fetal Bovine Serum.  For this experiment, prepare a Binding Buffer solution.  BB is DMEM-HIGH unsupplemented, plus 0.1% BSA.  Make sure this solution is sterile, through sterile filtration in a tissue culture hood.


2.     Prepare a solution of  104 cpm/ml 125I erythropoietin (or 125I leptin) in BB solution.


a.        (Fresh 125I erythropoietin comes in ~4 X 104 cpm/ml.  Use 2.5ml/10 ml BB)

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





1.         Aspirate the media.   Make sure to tilt the dish and place the aspirator tip in the corner, to avoid sucking up cells.  They are adhesive cells, but they can be blown off the plate if enough force is used.


2.         Add 1 ml of 104 125I erythropoietin BB solution ( or 1 ml of 125I leptin BB solution to each well.  Again, make sure not to pipet directly onto the cells, as the cells will lose their adherence.


3.         Leave the cells at room temperature for 1-2 hours.


4.         Aspirate the 125I erythropoietin BB solution or 125I leptin BB solution .


5.         Wash two times with 1ml of  non-sterile PBS.




1.         Solubilize with 1ml 2% SDS.  Pipet up and down to make sure that all the cells are off the plate. 


2.        Place the solubilized 293 cells into an 12X 75 mm test tube for gamma counting.   Place these cells into the #39 rack.


3.        Make sure to include 1 ml of  125I erythropoietin BB solution or 125I Leptin BB solution as a count test tube.


4.        Use program #39 on the gamma counter.




Last updated 11/4/1998 by Sarah Davis