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.

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

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), MCF-7/GFP (mock-transfected) and MCF-7/Erβ (transfected with full length ERb cDNA) cells by Western Blot. Detection of b-Actin was used as loading control in both assays. Lane 1, recombinant ERα or ERβ protein (Panvera, MD); Lane 2, negative control (RIPA buffer); Lane 3, MCF-7 wild-type; Lane 4, MCF-7/ERb; Lane 5, MCF-7/GFP.
Fig. 2: Western blots of the EGFR and two of its downstream proteins in non small cell lung cancer xenografts (7117 Lu). Tumour-bearing mice were treated with solvent or the EGFR- inhibitors Cetuximab or Erlotinib for 2 weeks. Lane 1 and 2, control group; lane 3 - 5, Cetuximab- treatment; lane 6 - 8, Erlotinib- treatment.
Fig. 3: Western Blots of components of the mTOR-pathway and cyclin D1 in 3366 breast carcinoma xenografts. Tumour-bearing animals were treated with solvent, tamoxifen (TAM), the rapamycin-derivative RAD001 or with the combination of both compounds for 7 days. Total protein was isolated from 4 randomly selected shock frozen tumour samples of each treatment group and analysed by SDS-PAGE following antibody incubation and luminescence detection (ECL).