Western blot screening for monoclonal antibodies against human separase

• Published in: Journal of immunological methods Vol. 274; no. 1; pp. 105 - 113
• Main Authors: Chestukhin, Anton, DeCaprio, James A.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 01.03.2003; Elsevier
• Subjects: Animals; Antibodies, immunoglobulins; Antibodies, Monoclonal - immunology; Antibody Specificity; Biological and medical sciences; Blotting, Western - methods; Carrier Proteins - genetics; Cell Cycle Proteins - genetics; Cell Cycle Proteins - immunology; Endopeptidases; Female; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Glutathione S-transferase; Glutathione Transferase - genetics; Humans; Hybridomas; Immunization; Maltose-binding protein; Maltose-Binding Proteins; Mice; Mice, Inbred BALB C; Miniblotter; Mitosis; Molecular immunology; Monoclonal antibodies; Recombinant Fusion Proteins - immunology; Separase; Slot blot; ORF; Mitosis; IP; GST; i.p; Maltose-binding protein; mAb; i.v; Slot blot; Miniblotter; HAT; MBP; Glutathione S-transferase; ELISA; Human; Peptidases; Enzyme; Cysteine endopeptidases; Cell cycle; Immunoblotting assay; Hydrolases; Monoclonal antibody
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/S0022-1759(02)00508-2

Separase is a cysteine protease that participates in separation of sister chromatids during mitosis. Human separase is a 230-kDa enzyme that is inhibited by binding to its protein inhibitor securin, specific phosphorylation, and subcellular localization. To further characterize human separase, we raised monoclonal antibodies specific against a C-terminal fragment of the protein. A critical step in monoclonal antibody production procedure is the primary screening of hybridoma supernatants. Here we report primary screening protocol utilizing Western blot analysis. The described screening protocol is carried out using fusion of a human separase fragment with two different purification tags, maltose-binding protein (MBP) and glutathione S-transferase (GST). Immunization by MBP-fusion was followed by primary screening with both MBP- and GST-separase fusions combined in the same preparation separated in SDS-PAGE. This highly sensitive screening approach reduced the number of positive signals by eliminating antibodies specific for the purification tag used in the immunization procedure. The described separase-specific antibodies were suitable for detection of endogenous separase in crude extracts, immunoprecipitation, and immunofluorescent cell staining experiments. The presented procedure is fast, reproducible and could be adopted as a primary screening scheme for a variety of protein antigens.