Il27 Antibody

Anti-IL-27/p28 antibody in western blot shows detection of recombinant mouse IL-27/p28. Recombinant protein (0.1 µg) was loaded on to an SDS-PAGE gel, and after separation, transferred to nitrocellulose. The expected band is approximately 26 kDa in size. The membrane was blocked with 1% BSA in TBST for 30 min at RT, followed by incubation with Biorbyt's Anti-IL-27/p28 antibody diluted 1:1000 in 1% BSA in TBST overnight at 4°C. After washes, the blot was reacted with secondary antibody HRP Goat anti-Rat IgG antibody (p/n orb347790) diluted 1:40000 in blocking buffer (p/n orb348637) for 30 min at RT.

Mouse peritoneal macrophages were grown in culture for 24 hours, stimulated with 10 ng/mL IFN gamma and 1 ug/ml LPS for 14 hours and incubated for 4 hours with Bredfeldin A. Cells were harvested, washed, aliquoted 1x106 cells per sample, and fixed and permeabilized according to a standard protocol. Samples were stained with biotinylated primary anti-mouse p28 antibody at (0.1-10 ug/ml primary antibody alongside negative controls of unstimulated cells and isotype controls. Cells were stained with 0.25 ug/ml rat anti-mouse CD107b conjugated Alexa Fluor 647 and PHYCOERYTHRIN Conjugated secondary at 1:100 and analyzed by flow cytometry. Stimulated cells showed increase PE staining (horizontal axis) when compared with unstimulated cells.