Protein Quantitation (iTRAQ)

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.

Request Form: iTRAQ Analysis

For more information please refer to PI publications:

 

Frequently Asked Questions

What amount of protein is required?

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.

What type of samples can be analysed?

Cell lysates, secreted protein samples, but not radioactive samples.

# Note: secreted samples must be in serum/plasma free media.

What is the maximum volume of sample?

The sample volume must be less than 250µL.

Can I use protease inhibitors?

Yes, Protease inhibitors will minimise protein degradation.

Is it important that the proteome you are looking at is in currently available databases?

Yes, as iTRAQ analysis will not work on samples from an unknown genome.

 

Please refer to our FAQs brochure for more details.

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