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Problems with protein loss iTRAQ??

Problems with protein loss iTRAQ?? - SDS-PAGE Gel Electrophoresis Forum

Problems with protein loss iTRAQ?? - SDS-PAGE Forum and Gel Electrophoresis Forum. Discuss the running of agarose gels, sds-page gels, and other gels including sequencing, gradient or tricine.


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Old 03-22-2012, 10:05 AM
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Default Problems with protein loss iTRAQ??



Hey guys,

I have such a problem: I prepared an exosome isolation from culture media and the aim is to perform iTRAQ MS on them. So I used a Amersham 2D-Clean-Up kit for protein precipitation on purified exosomes and I obtained very nice protein jelly-pellet, which I resuspended in iTRAQ dissolution buffer and proceeded to do silver stain gel with 10% of the sample. But apparently there was no protein separated because the 4 min staining did not reveal bands. This took some 3 hours with the sample still on ice when I recognized that I forgot to add 0.1% SDS which is included in the iTRAQ kit. So I added it immediately and proceeded with electrophoresis and staining again. But I still not succeeded in getting the protein. Is it possible that the protein was immediately degraded as soon as I dissolved it in dissolution buffer without SDS? In such case, shouldn't I still be able to see some bands??

Thanks for smart answers.
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