Recombinant Anti-Interferon gamma antibody [EPR23991-53] (ab267369)

False colour image of Western blot: Anti-Interferon gamma antibody [EPR23991-53] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab267369 was shown to bind specifically to Interferon gamma. A band was observed at 28 kDa in wild-type Jurkat cell lysates with no signal observed at this size in IFNG knockout cell line ab273746 (knockout cell lysate ab275521). To generate this image, wild-type and IFNG knockout Jurkat cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged.Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

Western blot: Anti-Interferon gamma antibody [EPR23991-53] staining at 1/1000 dilution; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab267369 was shown to bind specifically to Interferon gamma. A band was observed at 28 kDa in treated wild-type Jurkat cell lysates with no signal observed at this size in treated IFNG knockout cell line ab273746 (knockout cell lysate ab275521). To generate this image, wild-type and IFNG knockout Jurkat cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were HRP conjugated Goat anti-Rabbit (H+L) secondary antibody and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution. Blot was developed with an ultra-high sensitivity ECL reagent.

Flow cytometric analysis of 2% paraformaldehyde fixed 0.1% Tween-20 permeabilized Human peripheral blood mononuclear cell (PBMC) treated with cell stimulation cocktail (80nM PMA+1.34uM Ionomycin+10.6uM Brefeldin A+2uM Monensin) for 6 hours cells labelling Interferon gamma with ab267369 at 1/500 dilution (0.1ug)/ Right compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control. A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody. Cells were stained with anti-CD4 conjugated to Pacific blue. Fixed with 2% PFA for 10 min followed by intracellularly staining with rabbit IgG or ab267369.