Subcellular methods

Antigen and antibody generation

PrEST regions (Agaton C et al. (2003); Lindskog M et al. (2005)) are first amplified with RT-PCR from total RNA template pools with specific oligonucleotide primers for each PrEST. Amplicons are automatically processed with solid phase restriction, and ligated into the plasmid vector pAff8c (Larsson M et al. (2000)) where the human gene fragment is fused to a dual tag consisting of a hexahistidyl (His6) tag in frame with an immunopotentiating Albumin Binding Protein (ABP)-tag. After transformation into E. coli Rosetta(DE3), inserts are verified by DNA sequencing to omit clones with mutations and approved clones are single cell streaked. 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.

All proteins are expressed as His6ABP fusions in E. coli shake flask cultures upon induction with Isopropyl-B-D-Thiogalactopyranoside (IPTG). 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 Immobilized 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 following steps are performed on the ÄKTAxpress chromatography system enabling a high-throughput semi-automated process where captured antibodies are eluted by a low pH glycine buffer and automatically loaded onto a desalting column for buffer exchange. Antibodies are supplemented with 50% glycerol and 0.02% sodium azide for long-term storage at -20°C. 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-251 MG), human plasma depleted of IgG and HSA and whole tissue lysates from human liver and tonsil. A selection of the published antibodies, initially scored as uncertain in the standard WB panel, have been revalidated in a WB set-up comprising an over-expression lysate (VERIFY Tagged Antigen™, OriGene Technologies, Rockville, MD) as a positive control.