iTRAQ enables relative and absolute quantitation of protein and peptides by labeling samples with isotope encoded reporter ions.
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 strong cation exchange chromatography. Each cation exchange fraction is further analysed by reverse phase LC-MALDI. Results are compared with special software.
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 your proteome that you are looking at is in currently available databases?
Yes, as iTRAQ analysis will not work on samples from an unknown genome.
Delivery
20 working days