Recombinant Anti-Interferon regulatory factor 9/IRF-9 antibody [EPR24260-55] - BSA and Azide free (ab282125)

False colour image of Western blot: Anti-Interferon regulatory factor 9/IRF-9 antibody [EPR24260-55] 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, ab271043 was shown to bind specifically to Interferon regulatory factor 9/IRF-9. A band was observed at 48 kDa in treated wild-type HeLa cell lysates with no signal observed at this size in IRF9 CRISPR-Cas9 edited cell line ab266051 (CRISPR-Cas9 edited cell lysate ab258472). The band observed in the CRISPR-Cas9 edited lysate lane below 48 kDa is likely to represent a truncated form of Interferon regulatory factor 9/IRF-9. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and IRF9 CRISPR-Cas9 edited HeLa cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution 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.

False colour image of Western blot: Anti-Interferon regulatory factor 9/IRF-9 antibody [EPR24260-55] 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, ab271043 was shown to bind specifically to Interferon regulatory factor 9/IRF-9. A band was observed at 48 kDa in treated wild-type A549 cell lysates with no signal observed at this size in IRF9 CRISPR-Cas9 edited cell line ab267119 (CRISPR-Cas9 edited cell lysate ab258473). The band observed in the CRISPR-Cas9 edited lysate lane below 48 kDa is likely to represent a truncated form of Interferon regulatory factor 9/IRF-9. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and IRF9 CRISPR-Cas9 edited 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 (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

This data was developed using the same antibody clone in a different buffer formulation (ab271043). ab271043 staining Interferon regulatory factor 9 in wild-type A549 cells and IRF9 knockout A549 cells treated with interferon-a1 (ab48750) at 10 ng/ml for 16 hours. The cells were fixed with 4% paraformaldehyde (10 min) then permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab271043 at 0.4 μg/ml concentration and ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor^® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor^® 594) (ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).

This data was developed using the same antibody clone in a different buffer formulation (ab271043). ab271043 staining Interferon regulatory factor 9 in wild-type HeLa cells and IRF9 knockout HeLa cells treated with interferon-a1 (ab48750) at 10 ng/ml for 16 hours. The cells were fixed with 4% paraformaldehyde (10 min) then permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab271043 at 0.4 μg/ml concentration and ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor^® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor^® 594) (ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).

This data was developed using ab271043, the same antibody clone in a different buffer formulation. 

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

IRF9 expression is increased by IFN-alpha (PMID:18190617).

Exposure time: 15 seconds.

This data was developed using ab271043, the same antibody clone in a different buffer formulation. 

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

IFR9 can be degraded by the EV71-encoded 3C protease. A 48-kDa full length and 30-kDa cleaved IRF9 are observed. The molecular weight is consistent with what have been described in literature (PMID:21536800).

Exposure time: 3 minutes.

This data was developed using ab271043, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded HeLa (human cervix adenocarcinoma epithelial cell) cells (A) and HeLa cells treated by 10 ng/ml IFN for 18h (B) tissue labelling Interferon regulatory factor 9/IRF-9 with ab271043 at 1/2000 dilution (0.233 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Almost no staining on HeLa cells (A) and strongly positive staining on HeLa cells treated by 10 ng/ml IFN for 18h (B). The section was incubated with ab271043 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).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

This data was developed using ab271043, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Human spleen tissue labelling Interferon regulatory factor 9/IRF-9 with ab271043 at 1/2000 dilution (0.233 ug/ml) followed by a ready to use (Bond™ Polymer Refine Detection). Positive staining on human spleen. The section was incubated with ab271043 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).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

This data was developed using ab271043, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Human lung tissue labelling Interferon regulatory factor 9/IRF-9 with ab271043 at 1/2000 dilution (0.233 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on human lung (PMID: 30355451). The section was incubated with ab271043 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).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

This data was developed using ab271043, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Clear cell carcinoma of human kidney tissue labelling Interferon regulatory factor 9/IRF-9 with ab271043 at 1/2000 dilution (0.233 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on Clear cell carcinoma of human kidney (PMID: 30355451). The section was incubated with ab271043 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).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

This data was developed using ab271043, the same antibody clone in a different buffer formulation.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HeLa  (human cervix adenocarcinoma epithelial cell) cells labelling Interferon regulatory factor 9/IRF-9 with ab271043 at 1/50 dilution(9.3 ug/ml), followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/500 dilution (Green). Confocal image showing increased nuclear and weak cytoplasmic staining in HeLa cells treated with Interferon alpha-1 (10 ng/ml) for 16 h is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/500 dilution.

This data was developed using ab271043, the same antibody clone in a different buffer formulation.

Interferon regulatory factor 9/IRF-9 was immunoprecipitated from 0.35 mg treated HeLa whole cell lysate with ab271043 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab271043 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: HeLa (human cervix adenocarcinoma epithelial cell) treated with 10 ng/ml human IFN alpha 1 (ab48750) for 16 hours, whole cell lysate 10 ug

Lane 2: ab271043 IP in HeLa treated with 10 ng/ml human IFN alpha 1 (ab48750) for 16 hours whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab271043 in HeLa treated with 10 ng/ml human IFN alpha 1 (ab48750) for 16 hours whole cell lysate

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

Exposure time: 3 minutes.

This data was developed using ab271043, the same antibody clone in a different buffer formulation.

Interferon regulatory factor 9/IRF-9 was immunoprecipitated from 0.35 mg THP-1 (human monocytic leukemia monocyte) whole cell lysate with ab271043 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab271043 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: THP-1 whole cell lysate 10 ug

Lane 2: ab271043 IP in THP-1 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab271043 in THP-1 whole cell lysate

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

Exposure time: 3 minutes.