Anti-Caspase-8 antibody [E7]

• WB

Lab

Western blot - Anti-Caspase-8 antibody [E7] (AB32397)

Lanes 1 - 4 : Merged signal (red and green). Green - ab32397 observed at 55, 43/41 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab32397 was shown to specifically react with HAP1 + Staurosproin when HAP1 + Staurosproin knockout samples were used. Wild-type and HAP1 + Staurosproin knockout samples were subjected to SDS-PAGE. ab32397 and ab9484 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/500 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Caspase-8 antibody [E7] (ab32397) at 1/500 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

Wild-type HAP1 whole cell lysate treated with Staurosporin at 20 µg

Lane 3:

Caspase-8 knockout HAP1 whole cell lysate at 20 µg

Lane 4:

Caspase-8 knockout HAP1 whole cell lysate treated with Staurosporin at 20 µg

Predicted band size: 55 kDa

false

• WB

Supplier Data

Western blot - Anti-Caspase-8 antibody [E7] (AB32397)

Blocking buffer and concentration : 5% NFDM/TBST.

Diluting buffer and concentration : 5% NFDM /TBST.

All lanes:

Western blot - Anti-Caspase-8 antibody [E7] (ab32397) at 1/1000 dilution

Lane 1:

Jurkat cell lysate at 20 µg

Lane 2:

HeLa cell lysate at 20 µg

Lane 3:

IM-9 cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), HRP-conjugated at 1/1000 dilution

Predicted band size: 55 kDa

false

• WB

Lab

Western blot - Anti-Caspase-8 antibody [E7] (AB32397)

Lanes 1- 4 : Merged signal (red and green). Green - ab32397 observed at 55 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab32397 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. ab32397 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 500 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 [E7] (ab32397) at 1/500 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 [E7] (AB32397)

Western blot : Anti-CASP8 antibody [E7] (ab32397) staining at 1/500 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab32397 was shown to bind specifically to CASP8. A band was observed at 55 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in CASP8 knockout cell line. To generate this image, wild-type and CASP8 knockout HCT 116 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-Caspase-8 antibody [E7] (ab32397) at 1/500 dilution

Lane 1:

Wild-type HCT 116 cell lysate at 20 µg

Lane 2:

CASP8 knockout HCT 116 cell lysate at 20 µg

Lane 3:

Wild-type HeLa cell lysate at 20 µg

Lane 4:

CASP8 knockout HeLa cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

false

• WB

Unknown

Western blot - Anti-Caspase-8 antibody [E7] (AB32397)

All lanes:

Western blot - Anti-Caspase-8 antibody [E7] (ab32397) at 1/500 dilution

All lanes:

HeLa cell lysate at 10 µg

Predicted band size: 55 kDa

Observed band size: 55 kDa

false

• WB

Lab

Western blot - Anti-Caspase-8 antibody [E7] (AB32397)

Western blot : Anti-Caspase-8 antibody [E7] ab32397 staining at 1/500 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 3pc 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 [E7] (ab32397) at 1/500 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

Observed band size: 60 kDa,37 kDa

false