Anti-IRAK4 antibody [2H9]

• WB

Lab

Western blot - Anti-IRAK4 antibody [2H9] (AB119942)

Lane 1 : Wild-type MCF7 cell lysate 20 ug Lane 2 : IRAK4 knockout MCF7 cell lysate 20 ug Lane 3 : Wild-type THP-1 cell lysate 20 ug Lane 4 : IRAK4 knockout THP-1 cell lysate 20 ug Lane 5 : A549 cell lysate 20 ug Western blot : Anti-IRAK4 antibody [2H9] (ab119942) staining at 1/1000 dilution, shown in green; Rabbit Anti-GAPDH antibody [EPR16891] (ab181602) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab119942 was shown to bind specifically to IRAK4. A band was observed at 52 kDa in wild-type MCF7 cell lysates and wild-type THP-1 cell lysates with no signal observed at this size in IRAK4 knockout cell line. To generate this image, wild-type and IRAK4 knockout MCF7 cell lysates and knockoutTHP-1 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-Mouse IgG H&L 800CW and Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-IRAK4 antibody [2H9] (ab119942) at 1/1000 dilution

Lane 1:

Wild-type MCF7 cell lysate at 20 µg

Lanes 2 and 4:

Western blot - Human IRAK4 knockout THP-1 cell line (<a href='/en-us/products/cell-lines/human-irak4-knockout-thp-1-cell-line-ab281630'>ab281630</a>) at 20 µg

Lane 5:

A549 cell lysate at 20 µg

Secondary

Lanes 1 - 6:

Goat anti-Mouse IgG H&L 800CW at 1/20000 dilution

Lanes 1 - 6:

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

Observed band size: 52 kDa

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• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IRAK4 antibody [2H9] (AB119942)

ab119942, at 1/200 dilution, staining IRAK4 in paraffin-embedded Human lung cancer tissue by Immunohistochemistry with DAB staining.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-IRAK4 antibody [2H9] (AB119942)

ab119942 stained HepG2 cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab119942 at 1/100 dilution overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- mouse (ab96879) IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43μM.

• Flow Cyt

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Flow Cytometry - Anti-IRAK4 antibody [2H9] (AB119942)

Flow cytometric analysis of Hela cells using ab119942 at 1/200 dilution (blue) and negative control (red).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IRAK4 antibody [2H9] (AB119942)

ab119942, at 1/200 dilution, staining IRAK4 in paraffin-embedded Human kidney cancer tissue by Immunohistochemistry with DAB staining.

• WB

Lab

Western blot - Anti-IRAK4 antibody [2H9] (AB119942)

False colour image of Western blot : Anti-IRAK4 antibody [2H9] staining at 1/500 dilution, shown in green; Rabbit Anti-GAPDH antibody [EPR16891] (ab181602) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab119942 was shown to bind specifically to IRAK4. A band was observed at 55 kDa in wild-type THP-1 cell lysates with no signal observed at this size in IRAK4 knockout cell line ab281630 (knockout cell lysate ab282980). To generate this image, wild-type and IRAK4 knockout THP-1 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-Mouse IgG H&L (IRDye^® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed (ab216777) at 1/20000 dilution.

All lanes:

Western blot - Anti-IRAK4 antibody [2H9] (ab119942) at 1/500 dilution

Lane 1:

Wild-type THP-1 cell lysate at 20 µg

Lane 2:

IRAK4 knockout THP-1 cell lysate at 20 µg

Lane 3:

U937 cell lysate at 20 µg

Lane 4:

U-87 MG cell lysate at 20 µg

Observed band size: 55 kDa

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• ELISA

Unknown

ELISA - Anti-IRAK4 antibody [2H9] (AB119942)

ELISA using ab119942.

• WB

Unknown

Western blot - Anti-IRAK4 antibody [2H9] (AB119942)

All lanes:

Western blot - Anti-IRAK4 antibody [2H9] (ab119942) at 1/500 dilution

All lanes:

Recombinant IRAK4 protein

Predicted band size: 52 kDa

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• WB

Unknown

Western blot - Anti-IRAK4 antibody [2H9] (AB119942)

All lanes:

Western blot - Anti-IRAK4 antibody [2H9] (ab119942) at 1/500 dilution

Lane 1:

THP-1 cell lysate

Lane 2:

HeLa cell lysate

Lane 3:

K562 cell lysate

Lane 4:

MCF7 cell lysate

Lane 5:

RAW264.7 cell lysate

Lane 6:

Jurkat cell lysate

Lane 7:

COS-7 (African green monkey kidney fibroblast-like cell line) cell lysate

Predicted band size: 52 kDa

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