Thesis final - after defense-7 - Jacobs University

Chapter 2
2. Experimental approach
2.1. Materials
Yeast extract, peptone, dextrose, analytical grade buffer salts was purchased from Applichem
(Darmstadt, Germany). The protease inhibitor cocktail was from Sigma-Aldrich Chemie
GmbH (Steinheim, Germany). Acrylamide and dithiothreitol (DTT) were purchased from
Carl-Roth GmbH (Karlsruhe, Germany). The Bradford Protein Assay Kit was purchased from
Pierce Biotechnology Inc. (Rockford, IL, USA). ColorPlus pre-stained protein marker (New
England Biolabs, Frankfurt, Germany) and N, N, N, N-tetramethyl-ethylenediamine (TEMED)
were obtained from Serva (Heidelberg, Germany). The Toyopearl ®
adsorbents were
purchased from Tosoh Biosciences GmbH (Stuttgart, Germany). The C10/10
chromatography column, chromatographic materials Sepharose-Phenyl, Source-Phenyl and
pre-cast gels for electrophoresis were purchased from GE healthcare Europe GmbH (Munich,
Germany).
2.2. Experimental strategy
The experimental strategy has been presented in Figure 6, which started with the cultivation
of yeast cells followed by cells disruption. The cell proteome was collected after cell
disruption and subjected to chromatographic fractionation using several hydrophobic
adsorbents. The chromatographic fractions were collected in triplicate. The two dimensional
gels were performed for the 42 fractions obtained during chromatography. Several proteins
from each gel were selected for identification by MALDI-ToF-MS. Computational tools were
used to calculate several properties of the proteins. Finally, the physicochemical properties of
the proteins were correlated with the chromatographic behavior during HIC. All these
experimental steps have been explained sequentially in the following text.
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