Anti-IRAK-1 antibody [EPR26375-90]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IRAK-1 antibody [EPR26375-90] (AB302554)

Immunohistochemical analysis of paraffin-embedded A Wild-type HAP1 (H tissue labeling IRAK-1 with ab302554 at 1/100 (4.53 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Positive staining on (A) wild-type HAP1 cell pellet, no staining on (B) IRAK1 knockout HAP1 cell pellet. The section was incubated with ab302554 for 30 mins at room temperature.The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IRAK-1 antibody [EPR26375-90] (AB302554)

Immunohistochemical analysis of paraffin-embedded Human lung cancer an tissue labeling IRAK-1 with ab302554 at 1/100 (4.53 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Positive staining in human lung cancer, no staining in the adjacent tissue. The section was incubated with ab302554 for 30 mins at room temperature.The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IRAK-1 antibody [EPR26375-90] (AB302554)

Immunohistochemical analysis of paraffin-embedded Human colon carcinom tissue labeling IRAK-1 with ab302554 at 1/100 (4.53 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Positive staining in human colon carcinoma. The section was incubated with ab302554 for 30 mins at room temperature.The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• WB

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Western blot - Anti-IRAK-1 antibody [EPR26375-90] (AB302554)

Blocking and diluting buffer and concentration : Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBST The samples were run on a Bis-Tris gel.Lysates/proteins at 20 µg per lane. Performed under reducing conditions.False colour image of Western blot : Anti-IRAK1 antibody (ab302554) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red.In Western blot, ab302554 was shown to bind specifically to IRAK1. A band was observed at 80 kDa in wild-type HAP1 cell lysates with no signal observed at this size in IRAK1 knockout cell line. To generate this image, wild-type and IRAK1 knockout HAP1 cell lysates were analyzed. First, samples were run on an SDS-PAGE gel then transferred onto an immobilon-FL PVDF membrane. Membranes were blocked in in Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS 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 (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes:

Western blot - Anti-IRAK-1 antibody [EPR26375-90] (ab302554) at 1/1000 dilution

Lane 1:

Wild-type HAP1 (human chronic myelogenous leukemia near-haploid cell line), whole cell lysate 20 µg

Lane 2:

IRAK1 knockout HAP1 whole cell lysate 20 µg

Lane 3:

HeLa (human cervical adenocarcinoma epithelial cell), whole cell lysate 20 µg

Secondary

Lanes 1 - 3:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Lanes 1 - 3:

Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/10000 dilution

Observed band size: 80 kDa

false

• WB

Supplier Data

Western blot - Anti-IRAK-1 antibody [EPR26375-90] (AB302554)

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

This blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

All lanes:

Western blot - Anti-IRAK-1 antibody [EPR26375-90] (ab302554) at 1/500 dilution

All lanes:

Human hypothalamus tissue lysate 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/1000 dilution

Observed band size: 80 kDa

true

Exposure time: 59s

• WB

Supplier Data

Western blot - Anti-IRAK-1 antibody [EPR26375-90] (AB302554)

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

This blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

All lanes:

Western blot - Anti-IRAK-1 antibody [EPR26375-90] (ab302554) at 1/500 dilution

All lanes:

Human tonsil tissue lysate at 40 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 80 kDa

true

Exposure time: 180s