CELL FRACTIONATION AND QUANTITATION VIA LC-MS-MS
Posted: Fri Mar 29, 2013 2:55 pm
Hello all,
Has anybody ever performed quantitative analysis of cell fractionated material using LC-MS-MS.
Basically our goal is to separate out the cytoplasm from nuclei and we have performed this successfully using a kit from sigma.
We typically incubate our "cells" with drug medium for 12 hours and let the cells internalize our drug.
Then we seperate the components from the kit , and quantitate the fractions.
At the end we do get a result, obviously much higher in the cytoplasmic fraction compared to the nuclei fraction.
Does anybody know how to go about with a validation of this sort of method?
I am wondering if the final nuclei content in the pellet is indeed from the nuclei and not some of the sample that adhered to the polypropylene tubes.
Usually at the end we spike internal standard to the dried down fraction, since there are so many lipids we always filter the extracts before reconstitution in mobile phase and starting the run. We do observe much loss of analyte due to adsorbing to the filter, but luckily we have the internal standard which is also affected.
The kit we are using is the Nuclei Isolation Kit: Nuclei EZ Prep
The separation procedure is listed here
http://www.sigmaaldrich.com/etc/mediali ... 101bul.pdf
Any ideas, comments , or suggestions?
Thanks guys
Has anybody ever performed quantitative analysis of cell fractionated material using LC-MS-MS.
Basically our goal is to separate out the cytoplasm from nuclei and we have performed this successfully using a kit from sigma.
We typically incubate our "cells" with drug medium for 12 hours and let the cells internalize our drug.
Then we seperate the components from the kit , and quantitate the fractions.
At the end we do get a result, obviously much higher in the cytoplasmic fraction compared to the nuclei fraction.
Does anybody know how to go about with a validation of this sort of method?
I am wondering if the final nuclei content in the pellet is indeed from the nuclei and not some of the sample that adhered to the polypropylene tubes.
Usually at the end we spike internal standard to the dried down fraction, since there are so many lipids we always filter the extracts before reconstitution in mobile phase and starting the run. We do observe much loss of analyte due to adsorbing to the filter, but luckily we have the internal standard which is also affected.
The kit we are using is the Nuclei Isolation Kit: Nuclei EZ Prep
The separation procedure is listed here
http://www.sigmaaldrich.com/etc/mediali ... 101bul.pdf
Any ideas, comments , or suggestions?
Thanks guys