Anti-IRF5 抗体 [EPR17067]

• WB

Lab

Western blot - Anti-IRF5 antibody [EPR17067] (AB181553)

Lanes 1 - 2 : Merged signal (red and green). Green - ab181553 observed at 56 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab181553 was shown to specifically react with IRF5 in wild-type HAP1 cells as signal was lost in IRF5 knockout cells. Wild-type and IRF5 knockout samples were subjected to SDS-PAGE. ab181553 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 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-IRF5 antibody [EPR17067] (ab181553) at 1/1000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

IRF5 knockout HAP1 whole cell lysate at 20 µg

Predicted band size: 56 kDa

false

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IRF5 antibody [EPR17067] (AB181553)

Immunohistochemical analysis of paraffin-embedded Human spleen tissue labeling IRF5 with ab181553 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nuclear and cytoplasmic staining on leukocytes of Human spleen is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-IRF5 antibody [EPR17067] (AB181553)

Intracellular flow cytometric analysis of 2% paraformaldehyde-fixed THP-1 (Human monocytic leukemia cells) cells labeling IRF5with ab181553 at 1/50 dilution (red) compared with a rabbit monoclonal IgG isotype control (ab172730;black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (FITC) at 1/150 dilution was used as the secondary antibody.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IRF5 antibody [EPR17067] (AB181553)

Immunohistochemical analysis of paraffin-embedded Human lung cancer tissue labeling IRF5 with ab181553 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nucleus staining on cancer cells of Human lung cancer is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IRF5 antibody [EPR17067] (AB181553)

Anti-IRF5 antibody [EPR17067] (ab181553)
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human tonsil tissue labelling IRF5 with ab181553 at a dilution of 1 : 500. Heat mediated antigen retrieval was performed using AR9 antigen retrieval solution, and microwave treatment for 15 min at 20% power. Anti-Rabbit/Mouse HRP polymer (PerkinElmer Opal Polymer HRP Ms Plus Rb) was used as secondary antibody. Opal tyramide amplification was performed using Opal 520 fluorophore. Counterstained with DAPI stain. Image scanned with Vectra 3.0 and analyzed via Phenochart software.
This image was courteously provided by Dr. Houssein Abdul Sater, Georgia Cancer Center.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-IRF5 antibody [EPR17067] (AB181553)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized THP-1 (Human monocytic leukemia cells) cells labeling IRF5 with ab181553 at 1/1000 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 dilution (green). Confocal image showing cytoplasmic and weakly nuclear staining on THP-1 cells. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).
The negative controls are as follows :
-ve control 1 : ab181553 at 1/1000 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
-ve control 2 : ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/500 dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IRF5 antibody [EPR17067] (AB181553)

Immunohistochemical analysis of paraffin-embedded Human breast cancer tissue labeling IRF5 with ab181553 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nuclear and cytoplasmic staining on leukocyte of Human breast cancer stroma is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IRF5 antibody [EPR17067] (AB181553)

Immunohistochemical analysis of paraffin-embedded Rat spleen tissue labeling IRF5 with ab181553 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nuclear and cytoplasmic staining on lymphocytes of rat spleen is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-IRF5 antibody [EPR17067] (AB181553)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized RAW 264.7 (Mouse macrophage cells transformed with Abelson murine leukemia virus) cells labeling IRF5 with ab181553 at 1/1000 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 dilution (green). Confocal image showing cytoplasmic and weakly nuclear staining on RAW 264.7 cells. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).
The negative controls are as follows :
-ve control 1 : ab181553 at 1/1000 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
-ve control 2 : ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/500 dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IRF5 antibody [EPR17067] (AB181553)

Immunohistochemical analysis of paraffin-embedded Mouse liver tissue labeling IRF5 with ab181553 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nuclear staining on kupffer cells of mouse liver is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IP

Supplier Data

Immunoprecipitation - Anti-IRF5 antibody [EPR17067] (AB181553)

IRF5 was immunoprecipitated from 1mg of RAW 264.7 (Mouse macrophage cells transformed with Abelson murine leukemia virus) whole cell lysate with ab181553 at 1/150 dilution. Western blot was performed from the immunoprecipitate using ab181553 at 1/1000 dilution. Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG, was used as secondary antibody at 1/1500 dilution.

Lane 1 : RAW 264.7 whole cell lysate 10 μg (Input).

Lane 2 : ab181553 IP in RAW 264.7 whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab181553 in RAW 264.7 whole cell lysate.

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

All lanes:

Immunoprecipitation - Anti-IRF5 antibody [EPR17067] (ab181553)

Predicted band size: 56 kDa

false

• WB

Supplier Data

Western blot - Anti-IRF5 antibody [EPR17067] (AB181553)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-IRF5 antibody [EPR17067] (ab181553) at 1/2000 dilution

All lanes:

Ramos (Human Burkitt's lymphoma cell line) whole cell lysate at 10 µg

Secondary

All lanes:

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

Predicted band size: 106 kDa,113 kDa,193 kDa,27 kDa,56 kDa,91 kDa

Observed band size: 106 kDa,113 kDa,193 kDa,27 kDa,30 kDa,55 kDa

false

Exposure time: 1min

• WB

Lab

Western blot - Anti-IRF5 antibody [EPR17067] (AB181553)

Western blot : Anti-IRF5 antibody [EPR17067] (ab181553) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab181553 was shown to bind specifically to IRF5. A band was observed at 50-70 kDa in wild-type A549 cell lysates with no signal observed at this size in IRF5 knockout cell line. To generate this image, wild-type and IRF5 knockout A549 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-IRF5 antibody [EPR17067] (ab181553) at 1/1000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

IRF5 knockout A549 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

Observed band size: 50 kDa,70 kDa

false

• WB

Supplier Data

Western blot - Anti-IRF5 antibody [EPR17067] (AB181553)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-IRF5 antibody [EPR17067] (ab181553) at 1/50000 dilution

Lane 1:

THP-1 (Human monocytic leukemia cells) whole cell lysate at 10 µg

Lane 2:

A20 (Mouse reticulum cell sarcoma cell line) whole cell lysate at 10 µg

Secondary

All lanes:

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

Predicted band size: 56 kDa

Observed band size: 55 kDa

false

Exposure time: 1min

• WB

Supplier Data

Western blot - Anti-IRF5 antibody [EPR17067] (AB181553)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-IRF5 antibody [EPR17067] (ab181553) at 1/2000 dilution

Lane 1:

Mouse spleen lysate at 10 µg

Lane 2:

Rat spleen lysate at 10 µg

Secondary

All lanes:

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

Predicted band size: 21 kDa,22 kDa,46 kDa,56 kDa

Observed band size: 21 kDa,46 kDa,55 kDa

false

Exposure time: 30s

• WB

Supplier Data

Western blot - Anti-IRF5 antibody [EPR17067] (AB181553)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-IRF5 antibody [EPR17067] (ab181553) at 1/2000 dilution

Lane 1:

Mouse thymus lysate at 10 µg

Lane 2:

Rat thymus lysate at 10 µg

Secondary

All lanes:

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

Predicted band size: 56 kDa

Observed band size: 55 kDa

false

Exposure time: 1min

• WB

Supplier Data

Western blot - Anti-IRF5 antibody [EPR17067] (AB181553)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-IRF5 antibody [EPR17067] (ab181553) at 1/5000 dilution

All lanes:

RAW 264.7(Mouse macrophage cells transformed with Abelson murine leukemia virus ) whole cell lysate at 10 µg

Secondary

All lanes:

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

Predicted band size: 56 kDa

Observed band size: 55 kDa

false

Exposure time: 5s

• WB

Supplier Data

Western blot - Anti-IRF5 antibody [EPR17067] (AB181553)

Transfected lysate from abcam (Cat# : ab94265).

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-IRF5 antibody [EPR17067] (ab181553) at 1/5000 dilution

Lane 1:

Non-transfected HEK293T (Human epithelial cells from embryonic kidney) whole cell lysate at 10 µg

Lane 2:

IRF5 transfected HEK293T (Human epithelial cells from embryonic kidney) whole cell lysate at 10 µg

Secondary

All lanes:

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

Predicted band size: 56 kDa

Observed band size: 55 kDa

false

Exposure time: 1min

• WB

Supplier Data

Western blot - Anti-IRF5 antibody [EPR17067] (AB181553)

Blocking and diluting buffer and concentration : 5% NFDM/TBST In Western blot, anti-IRF5 antibody (ab181553) staining at 1/1000 dilution. Anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution.

All lanes:

Western blot - Anti-IRF5 (phospho S446) antibody [EPR26132-254] (<a href='/products/primary-antibodies/irf5-phospho-s446-antibody-epr26132-254-ab309088'>ab309088</a>) at 1/1000 dilution

Lane 1:

293T cells transfected with a human IRF5 expression vector containing a His-tag whole cell lysate (untreated membrane) at 20 µg

Lane 2:

293T cells transfected with a human IRF5 expression vector containing a His-tag whole cell lysate (phosphatase treated membrane) at 20 µg

Secondary

All lanes:

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

Observed band size: 56 kDa

false

Exposure time: 180s