iTRAQ technology coupled with 1D or 2D LC-MS/MS for multiplexing 4-8 samples simultaneously in a single experiment is available. iTRAQ enables relative and absolute quantitation of proteins and peptides by labeling samples with isotope encoded reporter ions. Differential expression of proteins of interest can be determined.
The protein sample is acetone precipitated, digested and labeled. Four (or eight) different samples can be labeled with iTRAQ reagents. Labeled peptides are first separated by High pH reverse phase chromatography with fraction concatenation. Each concatenated fraction is further analysed by LC-MS/MS. Results are compared using a specialised software.
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For more information please refer to PI publications:
The optimal amount for an iTRAQ experiment is 50µg tagged protein. Losses during sample cleanup can be considerable and are sample specific. We therefore recommend that 200-500µg of sample is provided; greater amounts will improve sensitivity for low abundant proteins.
Cell lysates, secreted protein samples, but not radioactive samples.
# Note: secreted samples must be in serum/plasma free media.
The sample volume must be less than 250µL.
Yes, Protease inhibitors will minimise protein degradation.
Yes, as iTRAQ analysis will not work on samples from an unknown genome.
Please refer to our FAQs brochure for more details.