Development of Med28 Specific Monoclonal Antibodies Laboratory for Tracing of Gene Function Yu Min A, Jin Gu Cho, Sang Gyu Parkp Laboratory for Tracing of Gene Function, College of Pharmacy, Ajou University, Suwon, Gyunggi-do, Korea. Abstract Introduction Med28 plays a role in transcription, signal transduction, and cell proliferation. The over-expression of med28 is associated with tumor progression in in vitro and in vivo models. Recently, it has been reported that the elevated expression of med28 is associated with poor outcome in women with breast cancer. The expression level of med28 in in vitro and in vivo was examined by using anti-rabbit polyclonal antibody in previous report. In this study, we report for the first time the generation and characterization of four monoclonal antibodies against med28 through immunoblotting, immunofluorescence microscopy, immunoprecipitation, and immunohistochemical analyses. These antibodies will be useful to detect med28 in in vitro and in vivo. PolⅡ TATA gene ACT DBD Med28 Mediator complex Cancer cells Proliferation and so on.. Med28 Liver Testis Functions Expression Methods Results 1. Generation of Recombinant Med28 Protein The expression of the med28 protein was induced by 0.1 mM IPTG. Then proteins were loaded to nickel affinity chromatography. Proteins were dialyzed against 1X PBS containing 20% glycerol. Figure 1. Purification of recombinant human med28 antigen. The samples were analyzed by Coomassie blue-stained SDS-PAGE. Fig 2. Western blot analysis using mouse monoclonal antibodies. Raw264.7 cells and HEK293 cells were transiently transfected with pcDNA3 empty vector or pcDNA3-myc-human med28, and blotted with the each indicated purified monoclonal antibodies. Fig 3. Immunoprecipitation analysis using mouse monoclonal antibodies. HEK293 cells were transiently transfected with pcDNA3 empty vector or pcDNA3-GFP-human med28. Purified IgG (2 μg) was incubated with protein extracts for 4 hr at 4 ℃, and incubated with protein G agarose for 2 hr at 4 ℃. 2. Generation of Monoclonal Antibodies To generate mouse monoclonal antibody, female BALB/C mice (13 weeks old) were immunized subcutaneously. The emulsion was produced by complete mixing of med28 protein (200 μg/200 μl) with equal volume of complete Freund adjuvant. The mouse serum antibody titers were assessed by indirect ELISA kit that was coated with 0.1 μg/well of med28 protein. 3. ImmunoFluorescence Microscopy The cells were incubated with purified MED28 antibody (5 μg/ml) at RT for 1 hr, and incubated with diluted 1:100 FITC conjugated goat anti-mouse. Fig 4. Immunofluorescence miscroscopic analysis in MCF-7 cells. MCF-7 cells were transiently transfected with pcDNA3-myc-human med28 (A). (B) For the detection of endogenous med28, cells were stained with purified mouse anti-med28 monoclonal IgG (1 μg/ml) as indicated. Fig. 5. Immunohistochemical staining analysis using human breast cancer tissues. Human breast cancer tissues were embedded into paraffin, and sectioned with 5 um. Cells were detected with the UltraView Universal DAB detection kit (Ventana) according to the manufacturer’s protocol. 4. ImmunoBlotting The membrane was blocked with TBS containing 5% skim-milk, 0.5% Tween-20 at RT for 1 hr, and incubated with primary antibody (1:10,000) diluted in TBS containing 1% skim-milk, 0.2% Tween-20 at RT for 2 hr. Then, membrane was incubated with secondary antibody (1:20,000) diluted in TBS containing 1% skim-milk, 0.2% Tween-20 at RT for 1 hr. 5. ImmunoHistoChemical Staining Cells were detected with the UltraView Universal DAB detection kit (Ventana) according to the manufacturer’s protocol. 6. ImmunoPrecipitation Cells were lysed with RIPA lysis buffer. Purified IgG (2 μg) was incubated with protein extracts for 4 hr at 4 ℃, and incubated with protein G agarose for 2 hr at 4 ℃. The samples were loaded into 15% SDS-PAGE. Discussion & Conclusion References Monoclonal anti-med28 antibodies detected exo/endogenous med28 protein. Localization of exo/endogenous med28 was obtained by IF and IHC staining. Med28 was located at both cytoplasm and nucleus. ☆ Monoclonal anti-med28 antibodies will be useful to expand med28 research area in in vitro and in vivo.☆ Liu C, Nguyen M. Biochem Biophys Res Commun 2002;290(1):602-612. Zhang L, Brooks MN. Clin Cancer Res 2004;10(10):3504-3508. Lu M, Brooks MN. Cancer Res 2005; 65(14):6159-6166. Yoon NK, Maresh EL. BMC Cancer 2010;10:335. Cho JG, Park SG. Hybridoma 2012;31(5):358-363.