- Quantitative Proteome 33
- Vortex the gel pieces for 15 minutes and spin down. Transfer the
supernatant to the mixture collection tube.
- The volume of 25 mM TEAB added to the gel pieces varies with the
volume of the gel pieces.
- Centrifuge the mixture collection tube at 13,000 g for 30 seconds.
Transfer the supernatant to the peptide collection tube.
- The mixture collection tube may contain small gel pieces. Transfer
the supernatant carefully to ensure no gel piece is simultaneously
transferred to the peptide collection tube. It is fine to leave a small
amount of supernatant in the mixture collection tube.
- Add 400μL 0.1% TFA to the gel pieces. Repeat steps 4 to 5.
- Add 600μL 50% ACN/0.1% TFA to the gel pieces. Repeat steps 4
to 5. - Add 600μL 100% ACN to the gel pieces. Vortex erectly for 15 minutes
until the gel pieces become white color and aggregated. Transfer the
supernatant to the mixture collection tube. - Add appropriate amount of 100% ACN to the mixture collection tube.
Vortex erectly for 15 minutes until the gel pieces become white color and
aggregated. Transfer the supernatant to the peptide collection tube. - Filter the solution in the peptide collection tube with a syringe filter of
- 2 μm. Collect the filtered solution in the final peptide tube.
- We recommend to collect only 1 mL solution in each final peptide
tube to avoid the solution from leaking out of the tube. Based on the
volume of solution used for gel extraction, there may be multiple final
peptide tubes.
- Dry the extracted peptides in final peptide tubes with centrifugal
evaporator.
- Be sure that no ACN is left in the tubes. Do not make the extracted
peptides too dry, or it will not be easy to redissolve the peptides. - Do not discard the gel pieces until finishing the whole experiment.
iTRAQ tag labeling of peptides
- Calculate the volume of iTRAQ Dissolution Buffer (supplied in the
iTRAQ Reagents Multiplex Kit) needed to be added into the dried
peptide to make it a final concentration of about 1− 1. 5 μg/μL based on
the initial amount of protein chosen.