Biology Reference
In-Depth Information
Stacking
2a
2b
1a
1b
1 cm
Resolving
Fig.
1
Schematic representation of a gel, in which loading, staining, and band
cutting for a high-performance proteomics analysis are represented. The band
pattern corresponds to
Chlamydomonas reinhardtii
cells grown in mixo-
trophic media
6. Wash the gel in distilled water for 30 min.
7. Cut the lanes into two gel pieces, aiming to divide the total
intensity of the band equally between them. Cut all of the lanes
in the same way. We recommend choosing one clear band as a
cutting reference to make the divisions of all of the lanes of the
experiment. Transfer gel pieces to individual 1.5 mL tubes. At
this point the gel pieces can be long term stored at −20 °C.
Before freezing cover the pieces with distilled water.
This procedure is an adaption of [
5
] with increased effort in gel
washing and digestion. Reduction and alkylation of proteins is not
needed using the protocol described above. Double digestion
(LysC-Trypsin) do not improve the fi nal results.
3.4 Protein Digestion,
Peptide Desalting and
Concentration
1. Chop the gel pieces with a scalpel over a glass plate. The fi nal
size of acrylamide pieces should be around 1 mm
3
. Glass plate
should be carefully cleaned after each gel piece. Avoid taking
too much gel without proteins.
2. Transfer the gel pieces to a 1.5 mL low binding tube and keep
them on water until all samples are processed.
3. Remove water and add 1 mL of 25 mM AmBic, incubate
15 min at 37 °C in a thermal shaker. Discard supernatant.
4. Remove water and add 1 mL of 25 mM AmBic/50 %
acetonitrile, incubate 15 min at 37 °C in a thermal shaker.
Discard supernatant.
3.4.1 Protein Digestion
