Recombinant Anti-IL-1 beta antibody [EPR23851-127] (ab254360)

ab254360 staining IL-1 beta in wild-type THP-1 cells and IL1B knockout THP-1 cells (ab273762) +/- TPA (80nM overnight) and LPS (100ng/ml, 6h). 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 ab254360 at 0.4μg/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).

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

Exposure time: 3 minutes

Blocking and diluting buffer and concentration: 3% NFDM/TBST

Nitrocellulose membrane was used in this blot.

 Exposure time: 70 seconds

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized RAW 264.7 cells labelling IL-1 beta with ab254360 at 1/50 (10.28 µg/ml) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green) Confocal image showing increased cytoplasmic and nuclear staining in RAW 264.7 cells treated with Lipopolysaccharide (100 ng/ml) for 4 h then together with Brefeldin A (1 µg/ml) for another 3 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 ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized THP-1(Human monocytic leukemia monocyte) treated with 80nM TPA for 16 hours, then 100ng/ml LPS for 3 hours and 300ng/ml BFA for another 3 hours (Right)/ Untreated control (Left) cells labelling IL-1 beta with ab254360 at 1/500 dilution (0.1ug). A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) treated with 100ng/ml LPS for 4 hours and 1ug/ml BFA for another 3 hours (Right)/ Untreated control (Left) cells labelling IL-1 beta with ab254360 at 1/500 dilution (0.1ug). A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized NR8383 (rat lung macrophage (alveolar)) treated with 100ng/ml LPS for 4 hours and 1ug/ml BFA for another 3 hours (Right)/ Untreated control (Left) cells labelling IL-1 beta with ab254360 at 1/50 dilution (1ug)/ Right and Left. A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.