Anti-IL-1 beta antibody [EPR23851-127]

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-IL-1 beta antibody [EPR23851-127] (AB254360)

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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - 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).

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-IL-1 beta antibody [EPR23851-127] (AB254360)

Flow cytometry staining of human peripheral blood mononuclear cells (PBMCs) (top) or PBMCs treated with 1µg/mL LPS and 1µg/mL BrefeldinA for 18 Hours (bottom), with ab254360 (right) or Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (left).

PBMCs were incubated for 30 min on ice in 1x PBS containing 10 µg/ml human IgG and 10 % normal goat serum to block FC receptors and non-specific protein-protein interactions. Cells were fixed and permeabilised with BD Cytofix/Cytoperm™ for 20 min, followed by addition of the antibody ab254360 or Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (1x 10⁶ in 100 µl at 0.2 μg/ml (1/2,500)) for 30 min at 22°C .

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C

Acquisition of >30000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter. Events were gated on live cells.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-IL-1 beta antibody [EPR23851-127] (AB254360)

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.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-IL-1 beta antibody [EPR23851-127] (AB254360)

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 Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-IL-1 beta antibody [EPR23851-127] (AB254360)

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.

• WB

Lab

Western blot - Anti-IL-1 beta antibody [EPR23851-127] (AB254360)

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

Compared with ab254360, ab283818 has higher affinity. For better using ab254360, we recommend loading higher amount of human/rat lysate or using lower antibody dilution.

Lanes 1 - 6:

Western blot - Anti-IL-1 beta antibody [EPR23851-127] (ab254360) at 1/1000 dilution

Lanes 7 - 12:

Western blot - Anti-IL-1 beta antibody [RM1009] (<a href='/en-us/products/primary-antibodies/il-1-beta-antibody-rm1009-ab283818'>ab283818</a>)

Lanes 1 and 7:

Untreated THP-1 (human monocytic leukemia monocyte) whole cell lysate at 20 µg

Lanes 2 and 8:

THP-1 treated with 100ng/ml lipopolysaccharide (LPS) for 3h whole cell lysate at 20 µg

Lanes 3 and 9:

Untreated RAW 264.7 (mouse abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

Lanes 4 and 10:

RAW 264.7 treated with 100 ng/ml LPS and 300 ng/ml Brefeldin A (<a href='/en-us/products/sample-preparation-kits/brefeldin-a-solution-1000x-ab193369'>ab193369</a>) for 4 hours, whole cell lysate at 20 µg

Lanes 5 and 11:

Untreated NR8383 (rat lung macrophage (alveolar)) whole cell lysate at 20 µg

Lanes 6 and 12:

NR8383 treated with 100 ng/ml LPS for 7 hours and 1 µg/ml Brefeldin A (<a href='/en-us/products/sample-preparation-kits/brefeldin-a-solution-1000x-ab193369'>ab193369</a>) for the last 3 hours, whole cell lysate at 20 µg

Observed band size: 17.5 kDa,28 kDa,31 kDa

false

Exposure time: 102s

• WB

Lab

Western blot - Anti-IL-1 beta antibody [EPR23851-127] (AB254360)

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

Nitrocellulose membrane was used in this blot.

Exposure time : 70 seconds

All lanes:

Western blot - Anti-IL-1 beta antibody [EPR23851-127] (ab254360) at 1/1000 dilution

Lane 1:

Untreated NR8383 (rat lung macrophage (alveolar)) whole cell lysate at 20 µg

Lane 2:

NR8383 treated with 100 ng/ml LPS for 7 hours and 300 ng/ml Brefeldin A (<a href='/en-us/products/sample-preparation-kits/brefeldin-a-solution-1000x-ab193369'>ab193369</a>) for the last 3 hours, whole cell lysate at 20 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 1/50000 dilution

Predicted band size: 30 kDa

Observed band size: 17.5 kDa,28 kDa,31 kDa

false