Protein isolation is performed from in vitro as well as in vivo samples. Adherent or suspension cells are harvested from culture flask in a special lytic buffer, tumour samples are homogenized in RIPA buffer both provided with protease/ phosphatase inhibitors. If required protein lysates can be differentiated into cytosolic or nuclear fraction.

We are able to detect a broad spectrum of phosphorylated and unphosphorylated proteins, using SDS-PAGE for separation of proteins and optimised staining protocols.

Exemplary targets that are investigated routinely in our laboratory: the estrogen receptors α and β [Fig. 1], the EGFR/ERK/STAT panel [Fig. 2], cell cycle associated proteins (p21Waf1/Cip1, p27Kip1, p16INK4A, cyclin A and D) as well as members of the mTor pathway (p70S6K, 4EBP1, mTor, Akt, PTEN) [Fig. 3].
 
We use human specific antibodies from Becton Dickinson, Santa Cruz, Cell Signaling, Acris, Sigma-Aldrich and Dako. For semi-quantitative analysis β-actin is monitored as internal control and used for the normalisation of relative protein expression. It is possible to verify detection of special targets on customer demands.
 

Fig. 1: Expression of ERα and ERβ protein. Both receptors were analysed in MCF-7 (wild type, lane 3), MCF-7/Erβ (transfected with full length ERb cDNA, lane 4) b-Actin was used as loading control. Lane 1, recombinant ERα or ERβ protein (Panvera, MD); Lane 2, negative control (RIPA buffer); (Behrens 2007 )
Fig. 2: Western blots of the EGFR and two of its downstream proteins in non small cell lung cancer xenografts (Fichtner 2008 ). Tumour-bearing mice were treated with solvent (lane 1+2) or the EGFR- inhibitors Cetuximab (lane 3-5) or Erlotinib (lane 6-8) for 2 weeks.

Fig. 3: Western Blots of components of the mTOR-pathway and cyclin D1 in 3366 breast carcinoma xenografts. (Behrens 2007 ).