- Quantitative Proteome 35
- Prepare a new 2 mL protein lobind tube. Spin down the labeled peptides
and combine the four samples of labeled peptides to the new tube.
- Do not combine the labeled peptides to one of the sample tube to
ensure the amount of peptides of each sample in the combined tube
is equal.
- Dry the labeled peptides with centrifugal evaporator.
- No need to make the sample too dry. Proceed to the next step when
the labeled peptides become as a small drop of brown liquid.
Large-scale experiment: protein quantification
- The amount of peptides required for iTRAQ labeling is 150μgof
peptides for each sample.
- Measure the concentration of peptides with Pierce BCA Protein Assay
Kit. - Transfer the appropriate amount of peptides to a new 2 mL protein
lobind tube. - The amount of peptides in different samples should be equal for
comparison. Adjust each sample to have equivalent amount and volume
with iTRAQ Dissolution Buffer. - Label the tube with the name of the sample and iTRAQ isobaric tag
number intended to label the sample. - Bring the iTRAQ Reagent vials supplied in the iTRAQ Reagents
Multiplex Kit to room temperature. - Add 70μL absolute ethanol supplied in the iTRAQ Reagents Multiplex
Kit to each iTRAQ Reagent vial. Vortex the mixture for 1 minute and
spin down. - Calculate the amount of iTRAQ Reagent needed to label each sample.
Each iTRAQ Reagent vial is able to label up to 75μgofpeptides.Be
sure that the percentage of ethanol in each sample mixed with iTRAQ
Reagent must be over 50%.
- Two vials of iTRAQ Reagent are required to label 150μgofpeptides
in each sample.
- Equal amount of peptides from different samples are labeled by adding
the calculated amount of iTRAQ Reagent 114, iTRAQ Reagent 115,
iTRAQ Reagent 116, or iTRAQ Reagent 117. - Cap and seal the 2 mL protein lobind tube containing sample with
parafilm carefully.