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Major Protein Loss! Why?


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#1 Carol Choong

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Posted 05 December 2010 - 07:59 PM

Dear all,

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?

Carol

#2 neuron

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Posted 06 December 2010 - 07:58 AM

Hi,

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 :unsure: . 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 :blink: ...because my experience with iTRAQ was not good, and I did it long back, so I've forgotten lot of things :huh: ...anywaz all the best :)

#3 Carol Choong

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Posted 07 December 2010 - 07:22 AM

Thanks a lot for the advise.I think the prob not only come from cation exchange but as well due to the SepPak used to desalt my sample. Anyway, I think I might try to desalt my sample with Ziptip. I am currently trying to desalt my sample through SepPak without SCX and will see what happen.

Thanks again.




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