Anti-Caspase-8 antibody [E6] - BSA and Azide free

• WB

Lab

Western blot - Anti-Caspase-8 antibody [E6] - BSA and Azide free (AB247233)

Lanes 1 and 2 : Merged signal (red and green). Green - ab32125 observed at 55 kDa. Red - loading control, ab8226, observed at 42 kDa.

ab32125 was shown to recognize Caspase-8 when Caspase-8 knockout samples were used, along with additional cross-reactive bands. Wild-type and Caspase-8 knockout samples were subjected to SDS-PAGE. ab32125 and ab8226 (loading control to beta actin) were diluted 1/3000 and 1/1000 and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10 000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-Caspase-8 antibody [E6] (<a href='/en-us/products/primary-antibodies/caspase-8-antibody-e6-ab32125'>ab32125</a>)

Lane 1:

Wild-type HAP1 cell lysate at 20 µg

Lane 2:

Caspase-8 knockout HAP1 cell lysate at 20 µg

Predicted band size: 55 kDa

false

• WB

Lab

Western blot - Anti-Caspase-8 antibody [E6] - BSA and Azide free (AB247233)

This data was developed using the same antibody clone in a different buffer formulation (ab32125).

Lanes 1- 4 : Merged signal (red and green). Green - ab32125 observed at 55 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab32125 was shown to react with Caspase-8 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab264958 (knockout cell lysate ab256857) was used. Wild-type HeLa and CASP8 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab32125 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 3000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Caspase-8 antibody [E6] (<a href='/en-us/products/primary-antibodies/caspase-8-antibody-e6-ab32125'>ab32125</a>) at 1/3000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

CASP8 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human CASP8 (Caspase-8) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-casp8-caspase-8-knockout-hela-cell-line-ab264958'>ab264958</a>)

Lane 3:

Jurkat cell lysate at 20 µg

Lane 4:

SH-SY5Y cell lysate at 20 µg

Predicted band size: 55 kDa

Observed band size: 55 kDa

false

• WB

Lab

Western blot - Anti-Caspase-8 antibody [E6] - BSA and Azide free (AB247233)

This data was developed using ab32125, the same antibody clone in a different buffer formulation.

Western blot : Anti-Caspase-8 antibody [E6] ab32125 staining at 1/3000 dilution, shown in green; Mouse anti GAPDH (ab8245) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 60 kDa in Wild-type A549 cell lysates with no signal observed at this size in CASP8 knockout A549 cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5pc 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 800CW & Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-Caspase-8 antibody [E6] (<a href='/en-us/products/primary-antibodies/caspase-8-antibody-e6-ab32125'>ab32125</a>) at 1/3000 dilution

Lane 1:

Wild-type A549 at 20 µg

Lane 2:

Western blot - Human CASP8 knockout A549 cell line (<a href='/en-us/products/cell-lines/human-casp8-knockout-a549-cell-line-ab286757'>ab286757</a>) at 20 µg

Lane 3:

Western blot - Human wild-type HCT116 cell line (ab288559) at 20 µg

Lane 4:

Western blot - Human CASP8 knockout HCT116 cell line (<a href='/en-us/products/cell-lines/human-casp8-knockout-hct116-cell-line-ab286576'>ab286576</a>) at 20 µg

Secondary

Lanes 1 - 4:

Goat anti-Rabbit 800CW at 1/20000 dilution

Lanes 1 - 4:

Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 60 kDa,37 kDa

Observed band size: 60 kDa

false