One common way to identify proteins in tissues or cells is through isolation by specific binders, often antibodies. To avoid false-positive results, not only the antibody specificity but also the washing and elution conditions are crucial.
Sophia Hober and co-workers, KTH (Royal Institute of Technology), Sweden report a competitive elution approach that uses monospecific antibodies to selectively pullout target proteins from liver extracts. The target proteins were competitively eluted from the antibody affinity media with the recombinant antigen. To remove the antigen from the eluted sample, IMAC spin columns were utilized to bind the N-terminal His-tag of the antigens. SDS-PAGE analysis and mass spectrometry confirmed affinity isolation of expected target proteins.
The new technique offers an effective approach for wide-scale pullout experiments where proteins and their interaction partners are studied.
Image: Staining of paraffin-embedded formalin-fixed liver using cTAGE5-specific antibodies indicates that cTAGE5 are present in the cytoplasm of hepatocytes. (c) Wiley-Blackwell
- Targeted protein pullout from human tissue samples using competitive elution
R. Falk, M. Ramström, C. Eriksson, M. Uhlén, H. Wernérus, S. Hober,
Biotechnol. J. 2011, 6.