Please make sure that blood was drawn at steady-state: withdraw food at 5:00 p.m.  and  draw blood no earlier than 12 noon the following day, to make sure that C-peptide reaches steady-state levels as it takes longer for the kidney to clear it. Calculate both insulin and C-peptide levels in pM. Then, make sure you use the mean of the ratio of C-peptide to insulin ratio of each mouse, not the ratio of the means. 

If you need a more sophisticated approach to measure clearance of 125I- Insulin injected via the tail vein, i can send you the protocol via email. However, i caution you that this is not straight-forward as injecting the trial vein of C57BL6 mice is not easy. It takes special surgical skill. Nonetheless, we have published this method. most recently in Al-Share QY, Diabetes 2015. Please let me know if you still need the protocol.

Best of luck 

Hi Sonia,

I'm very sorry but only today I saw your reply. I would very much appreciate if you could send me your protocol for the 125I-insulin injection via the tail vein, as we have already measured C-peptide and insulin and the ratio is altered.

I was thinking as well to measure IDE activity in the liver. By any chance, would you have a protocol? I found a kit from Merck-Millipore, but unfortunately, it has been discontinued.

Cheers