- Destaining solution shall be freshly prepared just before use.
- Freshly make the DTT solution from DTT powder.
- Freshly make the IAA solution from IAA powder.
- Always prepare the trypsin solution freshly from lyophilized
powder just before use. - Grow the main cultures without antibiotics.
- The filtered liquid can be stored in a 80 C freezer to con-
tinue the following procedure at a later time point. - Carefully discard the TCA containing supernatant into the
appropriate liquid waste container. - This acetone wash step is to remove the remaining TCA and
H 2 O, and repeat this step may increase the purity of the pre-
cipitated proteins. - Dried pellet looks sticky.
- The cell pellet can be stored at 80 C freezer for long.
- After this step, if the lysate still has sticky or snot-like lumps,
additional sonication may apply to break down the DNA in the
lysate. - We found 2μg protein is enough for the following process and
MS analysis. - Cutting each gel band further into few smaller pieces can
increase the efficiency of the following process. If not continue
the protocol right away, store the gel slices at 20 C. We
routinely use Protein LoBind 1.5 ml tubes (Eppendorf). - In this step and later procedures, unless stated otherwise, we
use 1 ml as the solution volume for each gel fraction/each
tube. - IAA is light sensitive.
- Acetonitrile dehydrates the gel slices. The gel slices shrink and
become white/opaque. - We add concentrated trypsin solution to the dried gel slices so
that the trypsin can be absorbed into the gel to increase the
digestion efficiency. - The final volume of each gel fraction/each tube is 665μl. The
final concentration of formic acid is 1%. - We usually use an LTQ-Orbitrap Velos mass spectrometer at
the Functional Genomic Centre Zurich (FGCZ).€
Acknowledgments
We thank Christian Ahrens (Agroscope W€adenswil & Swiss Insti-
tute of Bioinformatics SIB, Switzerland) for carefully reading and200 Yilei Liu et al.