Anti-FACL4 antibody [EPR8640] - Mouse IgG1 (Chimeric)

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-FACL4 antibody [EPR8640] - Mouse IgG1 (Chimeric) (AB322988)

ab322988 staining FACL4 in Hep G2 (positive) and MCF-7 (negative) cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab322988 at 1µg/ml (shown in green) and ab202272, Alexa Fluor® 594 Rabbit monoclonal [EP1332Y] to alpha Tubulin - Microtubule Marker (shown in pseudocolour magenta). Cells were then incubated with ab150117, Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution. Nuclear DNA was labelled with DAPI (shown in blue). Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• WB

Lab

Western blot - Anti-FACL4 antibody [EPR8640] - Mouse IgG1 (Chimeric) (AB322988)

False colour image of Western blot : Anti-FACL4 antibody [EPR8640] - Mouse IgG1 (Chimeric) (ab322988) staining at 1/1000 dilution, shown in black; Rabbit anti-GAPDH loading control staining, shown in magenta. In Western blot, ab322988 was shown to bind specifically to ACSL4. A band was observed at 79 kDa in Wild-type HeLa, HepG2 and Caco-2 cell lysates, with no signal observed at this size in Wild-type MCF7 cell lysates. A band of the same size was also observed in human kidney lysate and lower expression was observed in human heart lysate. Samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5 % 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-Mouse IgG Fc (HRP) preadsorbed (ab98717) at 1 : 10000 and Goat anti-Rabbit IgG H&L 680RD at 1 : 20000.

All lanes:

Western blot - Anti-FACL4 antibody [EPR8640] - Mouse IgG1 (Chimeric) (ab322988) at 1/1000 dilution

Lane 1:

Wild-type HelA cell lysate

Lane 2:

Wild-type HepG2 cell lysate

Lane 3:

Human Kidney lysate

Lane 4:

WIld-type Caco-2 cell lysate

Lane 5:

Wild-type MCF7 cell lysate

Lane 6:

Human heart lysate

Secondary

Lanes 1 - 6:

Western blot - Goat Anti-Mouse IgG Fc (HRP) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-fc-hrp-preadsorbed-ab98717'>ab98717</a>) at 1/10000 dilution

Lanes 1 - 6:

Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution

Observed band size: 79 kDa

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