重组Anti-IL-1 beta抗体[RM1009] (ab283818)

Western blot: Anti-IL1B antibody [RM1009] (ab283818) staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab283818 was shown to bind specifically to IL1B. A band was observed at 34 kDa in wild-type THP-1 cell lysates with no signal observed at this size in IL1B knockout cell line. To generate this image, wild-type and IL1B 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-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

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

Expression of IL-1 beta is induced by LPS treatment. 31-kDa precursor IL-1 beta, 28- and 17.5-kDa proteolytically cleaved IL-1 beta are observed.

The expression pattern and  molecular weight observed is consistent with what has been described in the literature (PMID: 8446594, 19559631)

Exposure time: Lane 1-3: 3 min ; Lane 4-7: 70 seconds

Immunohistochemical analysis of paraffin-embedded Mouse colon carcinom tissue labelling IL-1 beta with ab283818 at 1/500 (1.058 ug/ml) followed by a ready to use LeicaDS9800 (Bond™, Polymer Refine Detection). Positive staining on the lamina propria in mouse colon carcinoma. The section was incubated with ab283818 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 Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.

Immunohistochemical analysis of paraffin-embedded RAW 264.7 cells labelling IL-1 beta with ab283818 at 1/500 (1.058 ug/ml) followed by a ready to use LeicaDS9800 (Bond™, Polymer Refine Detection). Positive staining on RAW 264.7 cells treated with LPS (100ng/ml for 4+3 hours) added BFA (1µg/ml for 3 hours) (Image A) and no staining on control RAW 264.7 (Image B). The section was incubated with ab283818 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 Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins

Immunohistochemical analysis of paraffin-embedded Mouse lung tissue labelling IL-1 beta with ab283818 at 1/500 (1.058 ug/ml) followed by a ready to use LeicaDS9800 (Bond™, Polymer Refine Detection). Positive staining on mouse lung treated with LPS (1ug/ml for 16 hours) and BFA (1ug/ml for 16h hours) (Image A) and no staining on control mouse lung was observed (Image B). The section was incubated with ab283818 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 Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins

Immunohistochemical analysis of paraffin-embedded Mouse colon tissue labelling IL-1 beta with ab283818 at 1/500 (1.058 ug/ml) followed by a ready to use LeicaDS9800 (Bond™, Polymer Refine Detection). The section was incubated with ab283818 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.

Negative control: no staining on mouse colon.

Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™, Polymer Refine Detection).

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.

Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labelling IL-1 beta with ab283818 at 1/500 (1.058 ug/ml) followed by a ready to use LeicaDS9800 (Bond™, Polymer Refine Detection). The section was incubated with ab283818 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.

Negative control: no staining on mouse spleen.

Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™, Polymer Refine Detection).

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized U-937 cells labelling IL-1 beta with ab283818 at 1/50 (10.58 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2 ug/ml) dilution (Green). Confocal image showing cytoplasmic staining in U-937 cells treated with Phorbol-12-myristate-13-acetate (100 nM) for 2 days, then replaced it with Lipopolysaccharides (1 µg/ml) for 13 h, with the addition of Brefeldin A (5 µg/ml) for the last 4 hours. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 (2.5 ug/ml) 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 1/1000 (2 ug/ml) dilution.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized J774A.1 cells labelling IL-1 beta with ab283818 at 1/50 (10.58 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2 ug/ml) dilution (Green). Confocal image showing cytoplasmic staining in J774A.1 cells treated with Lipopolysaccharides (100 ng/ml) for 4h, with the addition of brefeldin A (1 ug/ml) for the last 3 hours. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 (2.5 ug/ml) 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 1/1000 (2 ug/ml) dilution.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized RAW 264.7 cells labelling IL-1 beta with ab283818 at 1/50 (10.58 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2 ug/ml) dilution (Green). Confocal image showing cytoplasmic and nuclear staining in RAW 264.7 cells transfected with Lipopolysaccharides (100 ng/ml) for 4h, with the addition of brefeldin A (1 ug/ml) for the last 3 hours. (PMID:18490713, PMID:18939951). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 (2.5ug/ml) 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 1/1000 (2 ug/ml) dilution.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized U-937 (Human histiocytic lymphoma monocyte) treated with 100nM PMA for 2days then 1µg/ml LPS treated for 13h and add 5µg/ml BFA for another 4h (Red) / U-937 treated with 100nM PMA for 2days (Green) cells labelling IL-1 beta with ab283818 at 1/500 dilution (0.1ug) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized J774A.1 (Mouse reticulum cell sarcoma monocyte macrophage) treated with 100ng/ml LPS treated for 4h and add 1µg/ml BFA for another 3h (Red) / Untreated control (Green) cells labelling IL-1 beta with ab283818 at 1/500 dilution (0.1ug) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

IL-1 beta was immunoprecipitated from 0.35 mg U-937 (Human histiocytic lymphoma monocyte) treated with 100nM PMA for 2 days, then 1µg/ml LPS for 13h and add 5µg/ml BFA for another 4 h, whole cell lysate 10ug with ab283818 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab283818 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: U-937 (Human histiocytic lymphoma monocyte) treated with 100nM PMA for 2 days, then 1µg/ml LPS for 13h and add 5µg/ml BFA for another 4 h, whole cell lysate 10ug

Lane 2: ab283818 IP in U-937 treated with 100nM PMA for 2 days, then 1µg/ml LPS for 13h and add 5µg/ml BFA for another 4 h, whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab283818 in U-937 treated with 100nM PMA for 2 days, then 1µg/ml LPS for 13h and add 5µg/ml BFA for another 4 h, whole cell lysate

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

IL-1 beta was immunoprecipitated from 0.35 mg RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) treated with 100ng/ml LPS for 4h and add 1ug/ml BFA for another 3 h whole cell lysate 10ug with ab283818 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab283818 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) treated with 100ng/ml LPS for 4h and add 1ug/ml BFA for another 3 h whole cell lysate 10ug

Lane 2: ab283818 IP in RAW 264.7 treated with 100ng/ml LPS for 4h and add 1ug/ml BFA for another 3 h whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab283818 in RAW 264.7 treated with 100ng/ml LPS for 4h and add 1ug/ml BFA for another 3 h whole cell lysate

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