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Amplicons are automatically processed with solid phase restriction, and ligated into the plasmid vector pAff8c Larsson M et al. After transformation into E. Plasmids are collected from all purified clones for deposition in the clone library and glycerol stocks are prepared and used as starting material for protein production.

A fully automated protein purification system has been developed to allow for purifications of up to 60 cell lysates at a time. One-step purification is enabled by the hexahistidine affinity tag and metal affinity chromatography IMAC and performed under denaturing conditions. After evaluation of protein concentration and purity, the molecular weight of the PrEST proteins is determined by mass spectrometry as a final quality control.

The purified proteins are then used to prepare antigens and affinity columns with PrEST-ligands. In addition, affinity resin with His6ABP-ligand is also produced. After immunization of the antigens the polyclonal antisera, generated together with collaborative partners, are carefully purified in a three-step fashion consisting of: depletion of unwanted specificity, capture of wanted specificity and a final buffer exchange step.

A manual process using gravity-flow columns carries out depletion of antibodies with unwanted specificity. The binding specificity of all antibodies is determined on protein microarrays to certify that only antibodies with high specificity and low background binding are approved for immunohistochemistry analysis. All approved antibodies are further analyzed in a high-throughput WB platform using protein lysates from human cell lines RT-4 and U MG , human plasma depleted of IgG and HSA and whole tissue lysates from human liver and tonsil.