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- Posts: 15
- Joined: Wed Jan 12, 2011 10:13 am
Dear all,
So far, I have been using protein precipitation protocols (mostly, stepwise addition of 3:1 icecold acetonitrile:biological fluid) with great success (peptide recovery > 80%)
This time, however, I cannot get a satisfying human plasma recovery for the 15peptide I am studying (not greater than 60%).
I have tested a variety of conditions; icecold ethanol against acetonitrile, centrifugation at room temperature against 4C, leaving samples with organic precipitant for 20 min before centrifuge or not, filter sample or not. The "best" I could get was a recovery from human plasma of 60 %, following stepwise addition of icecold ethanol(!!) - while every single paper admits acetonitrile is the best organic to use - vortex, centrifuge (10min, 15000 rpm, 4C), filter.
I am using a UPLC method for detection.
Any feedback/idea/suggestion would be very much appreciated!!
Kind Regards,
So far, I have been using protein precipitation protocols (mostly, stepwise addition of 3:1 icecold acetonitrile:biological fluid) with great success (peptide recovery > 80%)
This time, however, I cannot get a satisfying human plasma recovery for the 15peptide I am studying (not greater than 60%).
I have tested a variety of conditions; icecold ethanol against acetonitrile, centrifugation at room temperature against 4C, leaving samples with organic precipitant for 20 min before centrifuge or not, filter sample or not. The "best" I could get was a recovery from human plasma of 60 %, following stepwise addition of icecold ethanol(!!) - while every single paper admits acetonitrile is the best organic to use - vortex, centrifuge (10min, 15000 rpm, 4C), filter.
I am using a UPLC method for detection.
Any feedback/idea/suggestion would be very much appreciated!!
Kind Regards,
