Anti-FADD antibody [EPR4415]

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-FADD antibody [EPR4415] (AB108601)

Intracellular Flow Cytometry analysis of A431 (human epidermoid carcinoma) cells labeling FADD with purified ab108601 at 1/140 dilution (10ug/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488) (1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) was used as the unlabeled control.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-FADD antibody [EPR4415] (AB108601)

Immunohistochemical staining of paraffin-embedded Human kidney tissue using ab108601 at a dilution of 1/100.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• IP

Lab

Immunoprecipitation - Anti-FADD antibody [EPR4415] (AB108601)

FADD was immunoprecipitated from 0.35 mg HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10 μg with 108601 at 1/120 dilution (2μg). VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10 μg

Lane 2 : ab108601 IP in HeLa whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab108601 in HeLa whole cell lysate

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-FADD antibody [EPR4415] (ab108601)

Predicted band size: 23 kDa

Observed band size: 23 kDa

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• WB

Lab

Western blot - Anti-FADD antibody [EPR4415] (AB108601)

Lanes 1 - 4 : Merged signal (red and green). Green - ab108601 observed at 25 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab108601 was shown to specifically react with FADD when FADD knockout samples were used. Wild-type and FADD knockout samples were subjected to SDS-PAGE. ab108601 and ab8245 (loading control to GAPDH) were diluted 1/1000 and 1/2000 respectively 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-FADD antibody [EPR4415] (ab108601) at 1/1000 dilution

Lane 1:

Wild-type HAP1 cell lysate at 20 µg

Lane 2:

FADD knockout HAP1 cell lysate at 20 µg

Lane 3:

A431 cell lysate at 20 µg

Lane 4:

Jurkat cell lysate at 20 µg

Predicted band size: 23 kDa

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• WB

Lab

Western blot - Anti-FADD antibody [EPR4415] (AB108601)

Lanes 1 - 2 : Merged signal (red and green). Green - ab108601 observed at 23 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab108601 was shown to react with FADD in wild-type HeLa cells in western blot with loss of signal observed in FADD knockout cell line ab261817 (FADD knockout cell lysate ab257261). Wild-type and FADD knockout HeLa cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab108601 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated 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 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-FADD antibody [EPR4415] (ab108601) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

FADD knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human FADD knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-fadd-knockout-hela-cell-line-ab261817'>ab261817</a>)

Predicted band size: 23 kDa

Observed band size: 23 kDa

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• WB

Unknown

Western blot - Anti-FADD antibody [EPR4415] (AB108601)

All lanes:

Western blot - Anti-FADD antibody [EPR4415] (ab108601) at 1/1000 dilution

Lane 1:

A431 cell lysate at 10 µg

Lane 2:

Jurkat cell lysate at 10 µg

Lane 3:

HeLa cell lysate at 10 µg

Lane 4:

SKBR-3 cell lysate at 10 µg

Predicted band size: 23 kDa

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