Major Protein Loss! Why?
Posted 05 December 2010 - 07:59 PM
I am now currently dealing with serum samples. First I deplete albumin and IgG from my samples (by using Calbiochem depletion kit). Then I digest my samples with trypsin and subject to LC-MAlDI and MALDI-ToF/ToF. There are about 110 proteins found.
Then I proceed, where I carry out ABI iTRAQ Cation Exchange chromatography (SCX) and clean my eluted samples (from SCX) by using C18 SepPak Column. Then my protein found are only 65. Anyone experience the same thing before? How can I overcome it?
Posted 06 December 2010 - 07:58 AM
I am not perfect in using iTRAQ but since I've done it long ago , I know u loose lot of protein when u do the cation exchange. Actually in any coulmn or precipitation u loose some amount of protein but this much loss, even I don't know . what if u don't do the cation exchange, and only desalt ur sample with ziptip???...well I am not sure weather this would help ...because my experience with iTRAQ was not good, and I did it long back, so I've forgotten lot of things ...anywaz all the best
Posted 07 December 2010 - 07:22 AM