I am measuring hydroxyproline in mouse pulmonary arteries. I have tried to hydrolyze collagen by 2N HCl and by 2N NaOH, and I found the use of 2N NaOH can improve the read-out. The result using testing arteries seems OK.

But after using the experimental arteries, in the group that should have less collagen, I got very high concentration reading; whereas in the other group that should have more collagen, the measured concentration is low! Exactly the opposite result than I expected.

The standard curve was great so no problem should be with the Chloramine T or pDAB solutions.

What could go wrong?

Thanks!