Anti-c-Fos antibody [EPR883(2)]

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-c-Fos antibody [EPR883(2)] (AB134122)

Overlay histogram showing HeLa cells stained with ab134122 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab134122, 1/10000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

• WB

Lab

Western blot - Anti-c-Fos antibody [EPR883(2)] (AB134122)

False colour image of Western blot : Anti-c-Fos antibody [EPR883(2)] staining at 1/1000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab134122 was shown to bind specifically to c-Fos. A band was observed at 45-55 kDa in treated wild-type PC-12 cell lysates with no signal observed at this size in FOS knockout cell line ab281615 (knockout cell lysate ab283111). Treatment of NGF has no observable affect on protein expression in this cell line. To generate this image, wild-type and FOS knockout PC-12 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 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-c-Fos antibody [EPR883(2)] (ab134122) at 1/1000 dilution

Lane 1:

Wild-type PC-12 Untreated Control NGF (0 ng/mL, 4 days) cell lysate at 20 µg

Lane 2:

Wild-type PC-12 Treated NGF (111 ng/mL, 4 days) cell lysate at 20 µg

Lane 3:

FOS knockout PC-12 Untreated Control NGF (0 ng/mL, 4 days) cell lysate at 20 µg

Lane 4:

FOS knockout PC-12 Treated NGF (111 ng/mL, 4 days) cell lysate at 20 µg

Observed band size: 45-55 kDa

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• WB

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Western blot - Anti-c-Fos antibody [EPR883(2)] (AB134122)

All lanes:

Western blot - Anti-c-Fos antibody [EPR883(2)] (ab134122) at 1/1000 dilution

Lane 1:

Lysate of U937 cells treated with TPA at 10 µg

Lane 2:

U937 cell lysate at 10 µg

Lane 3:

Lysate of HeLa cells treated with TPA at 10 µg

Lane 4:

HeLa cell lysate at 10 µg

Secondary

All lanes:

Goat ant-rabbit HRP conjugated antibody at 1/2000 dilution

Predicted band size: 41 kDa

Observed band size: 62 kDa

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