Charles University in Prague - 2nd Faculty of Medicine, V Úvalu 84, Praha 5, 150 06
Multiplexed immuno-precipitation with 1725 commercially available antibodies to cellular proteins.
Slaastad H, Wu W, Goullart L, Kanderova V, Tjønnfjord G, Stuchly J, Kalina T, Holm A, Lund-Johansen F. Proteomics. 2011 Dec;11(23):4578-82. doi: 10.1002/pmic.201000744. Epub 2011 Nov 7. IF: 4.815
Department of Paediatric Haematology and Oncology
Antibody array analysis of complex samples requires capture reagents with exceptional specificity. The frequency of antibodies with label-based detection may be as low as 5%. Here, however, we show that as many as 25% of commercially available antibodies are useful when biotinylatedcellular proteins are fractionated by size exclusion chromatography (SEC) first. A microsphere multiplex with 1725 antibodies to cellular proteins was added to 24 SEC fractions, labelled with streptavidin and analyzed by flow cytometry (microsphere-based affinity proteomics, MAP). The SEC-MAP approach resolved different targets captured by each antibody as reactivity peaks across the separation range of the SEC column (10 - 670 kDa). Complex reactivity profiles demonstrated that most antibodies bound more than one target. However, specific binding was readily detected as reactivity peaks common for different antibodies to the same protein. We optimized sample preparation and found that amine-reactive biotin rarely inhibited antibody binding when the biotin to lysine ratio was kept below 1:1 during labelling. Moreover, several epitopes that were inaccessible toantibodies in native proteins were unmasked after heat denaturation with 0.1% of SDS. The SEC-MAP format should allow researchers to buildmultiplexed assays with antibodies purchased for use in e.g. Western blotting.
Submitted by , Department of Immunology, for posting on the web 6. 8. 2012.