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Dr Henrik Wernérus
Pia Angelidou (Research engineer), Cecilia Eriksson (PhD student), Martina Jones (Research engineer), Josefine Karlsson (technician), Karin Larsson (PhD student) and Cajsa Älgenäs (technician).
(i) Management of the antibody generation outsourcing, (ii) Generation of monospecific antibodies through affinity purification of polyclonal antisera, (iii) western blot analysis of antibodies approved in protein arrays, (iv) storage and distribution of antibodies within the program and to collaborative partners.
The Immunotechnology group is responsible for the generation, purification and Western blot analysis of all monospecific antibodies produced in the Human Protein Atlas (HPA) program.
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. Depletion of antibodies with unwanted specificity is carried out by a manual process using gravity-flow columns. 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. Following purification the antibodies are supplemented with 50% glycerol and 0,02% sodium azide for long-term storage at -20 C.
The binding specificity of all monospecific antibodies are determined on protein microarrays (Module 6) to certify that only antibodies with high specificity and low background binding are approved for immunohistochemistry analysis (Module 7). All approved antibodies are further analyzed by high-throughput western blot using a standard 1:250 dilution and protein lysates from human cell lines (RT-4 and U251mg), human plasma depleted of IgG and HSA and whole tissue lysates from human liver and tonsil, respectively.
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