Multiplex western blot analysis

For high-content Western analysis, DigiWest was performed as described by Treindl et al. [15]. In brief, xenograft tissue was ground by a Mikro-Dismembrator (Sartorius) and subsequently lysed by the addition of 2× LDS lysis buffer (Life Technologies^™) and heated to 95°C for 9 minutes with several vortexing steps. Undissolved material was removed by consecutive centrifugation at 20°C for 2 minutes at 300 x g, 5 minutes at 16,000 x g, and through a QIAshredder homogenizer tube (Qiagen) for 5 minutes at 16,000 x g. Protein concentration was determined by in-gel staining. 3 μL lysate was separated by SDS-PAGE stained with IRDye^® Blue and detected with a Li-COR Odyssey Infrared Imaging System (Li-COR Biosciences, Bad Homburg, Germany) at 700 nm. Next, 12 μg protein per sample was separated using 4–12% Bis-Tris gels (Life Technologies™) according to the manufacturer’s instructions. Blotting onto PVDF membranes (Millipore) was performed under standard conditions. Proteins immobilized on the blotting membrane were biotinylated (NHS-PEG12-Biotin, Thermo Scientific) and individual sample lanes were cut into 96 strips each (height 0.5 mm each, strip width 7.5 mm) using an electronic cutting tool (Silhouette SD). Each individual strip was placed in a separate well of a 96-well plate, and protein was eluted for 2 hours in 10 μL elution buffer (8 M urea, 1% Triton-X100 in 100 mM Tris-HCl, pH 9.5). After the addition of 90 μL of dilution buffer (5% BSA in PBS, 0.02% sodium azide, 0.05% Tween-20), 96 different Neutravidin-coated Luminex bead sets (40,000 beads/well) were added to the individual wells, and eluted biotinylated proteins were captured on the bead surface during overnight incubation. After incubation, the Luminex beads were pooled, washed, and stored in storage buffer (1% BSA, 0.05% Tween-20, 0.05% sodium azide in PBS) at 4°C.

For antibody incubation, an aliquot of the bead pool was transferred into an assay plate and 30 μL of diluted western blot antibody in assay buffer (Blocking Reagent, Roche Applied Science; 0.05% Tween 20, 0.02% sodium azide, 0.2% milk powder) was added per well. 116 antibody incubations on 12 samples were performed overnight at 4°C. For read-out, beads were washed twice with 100 μL of PBST before species-specific PE-labeled secondary antibodies (Jackson) were added in 30 μL of assay buffer for 1 hour. After two washes with 100 μL of PBST, the signal was generated in a FlexMAP 3D instrument (Luminex).