Anti-Interferon gamma antibody [EPR23991-53] - BSA and Azide free

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-Interferon gamma antibody [EPR23991-53] - BSA and Azide free (AB280574)

This data was developed using ab267369, the same antibody clone in a different buffer formulation.

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.

• WB

Lab

Western blot - Anti-Interferon gamma antibody [EPR23991-53] - BSA and Azide free (AB280574)

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.

All lanes:

Western blot - Anti-Interferon gamma antibody [EPR23991-53] (<a href='/en-us/products/primary-antibodies/interferon-gamma-antibody-epr23991-53-ab267369'>ab267369</a>) at 1/1000 dilution

Lane 1:

Wild-type Jurkat Vehicle control: PMA (0 ng/mL, 6 h) and ionomycin (0 µg/mL, 6 h), BFA (5 µg/ml, last 5 h) cell lysate at 20 µg

Lane 2:

Wild-type Jurkat Treated: PMA (25 ng/mL, 6 h) and ionomycin (1 µg/mL, 6 h), BFA (5 g/ml, last 5 h) cell lysate at 20 µg

Lane 3:

IFNG knockout Jurkat Vehicle control: PMA (0 ng/mL, 6 h) and ionomycin (0 µg/mL, 6 h), BFA (5 µg/ml, last 5 h) cell lysate at 20 µg

Lane 4:

IFNG knockout Jurkat Treated: PMA (25 ng/mL, 6 h) and ionomycin (1 µg/mL, 6 h), BFA (5 µg/ml, last 5 h) cell lysate at 40 µg

Predicted band size: 19 kDa

Observed band size: 28 kDa

false

• WB

Lab

Western blot - Anti-Interferon gamma antibody [EPR23991-53] - BSA and Azide free (AB280574)

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.

All lanes:

Western blot - Anti-Interferon gamma antibody [EPR23991-53] (<a href='/en-us/products/primary-antibodies/interferon-gamma-antibody-epr23991-53-ab267369'>ab267369</a>) at 1/1000 dilution

Lane 1:

Wild-type Jurkat Treated: PMA (25 ng/mL, 6 h) and ionomycin (1 μg/mL, 6 h), BFA (5 µg/ml, last 5 h) cell lysate at 40 µg

Lane 2:

IFNG knockout Jurkat Treated: PMA (25 ng/mL, 6 h) and ionomycin (1 µg/mL, 6 h), BFA (5 µg/ml, last 5 h) cell lysate at 40 µg

Lane 3:

PTA-6967 Vehicle control + Brefeldin A (5 ug/ml, 6 h) cell lysate at 10 µg

Lane 4:

PTA-6967 Treated TPA (80 nM, 5 h), Ionomycin <a href='/en-us/products/biochemicals/ionomycin-ca2-salt-ca2-ionophore-ab120116'>ab120116</a> (3 µM, 5 h) + Brefeldin A (5 µg/mL, 3 h) cell lysate at 10 µg

Predicted band size: 19 kDa

Observed band size: 28 kDa

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