Anti-IL-1 beta antibody [EPR21086]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-IL-1 beta antibody [EPR21086] (AB216995)

Immunohistochemical analysis of 4% paraformaldehyde fixed, 0.1% TritonX-100 permeabilised U-937 (human histiocytic lymphoma monocyte) cells labeling IL-1 beta with ab216995 at 1/50 (10 μg/ml) dilution. Followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed secondary antibody at 1/1000 (2 μg/ml) dilution. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain at 1/200 (2.5 μg/ml) dilution. Nuclear staining : DAPI.

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 addition of Brefeldin A (5 μg/ml) for the last 4 hours.

• IP

Unknown

Immunoprecipitation - Anti-IL-1 beta antibody [EPR21086] (AB216995)

IL-1 beta was immunoprecipitated from 0.35 mg THP-1 (human monocytic leukemia monocyte) whole cell lysate treated with 100 ng/ml lipopolysaccharide (LPS) for 3 hours, with ab216995 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab216995 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5000 dilution.

Lane 1 : THP-1 (human monocytic leukemia monocyte) treated with 100 ng/ml lipopolysaccharide (LPS) for 3 hours whole cell lysate 10 μg (Input).
Lane 2 : ab216995 IP in THP-1 treated with 100 ng/ml lipopolysaccharide (LPS) for 3 hours whole cell lysate (+).
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab216995 in THP-1 treated with 100 ng/ml lipopolysaccharide (LPS) for 3 hours whole cell lysate (-).
Blocking and dilution buffer and concentration : 5% NFDM/TBST.
Exposure time : 3 minutes.

All lanes:

Immunoprecipitation - Anti-IL-1 beta antibody [EPR21086] (ab216995)

Predicted band size: 30 kDa

false

• WB

Unknown

Western blot - Anti-IL-1 beta antibody [EPR21086] (AB216995)

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

The expression profile is consistent with the literature (PMID 15192144 and 10845914).

All lanes:

Western blot - Anti-IL-1 beta antibody [EPR21086] (ab216995) at 1/1000 dilution

Lane 1:

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

Lane 2:

THP-1 treated with 100 ng/ml Lipopolysaccharide (LPS) for 3 hours 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 1/100000 dilution

Predicted band size: 30 kDa

true

Exposure time: 3min

• WB

Lab

Western blot - Anti-IL-1 beta antibody [EPR21086] (AB216995)

Lanes 1 - 4 : Merged signal (red and green). Green - ab216995 observed at 27-32 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55 kDa.

ab216995 was shown to react with IL-1 beta in LPS-treated wild-type THP-1 cells in Western blot with loss of signal observed in IL1B knockout cell line ab273762 (LPS-treated) (knockout cell lysate ab275508). Wild-type THP-1 and IL1B knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab216995 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4 °C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated 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 in 20000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-IL-1 beta antibody [EPR21086] (ab216995) at 1/1000 dilution

Lane 1:

Wild-type THP-1 untreated cell lysate at 30 µg

Lane 2:

Wild-type THP-1 LPS-treated (3 h, 100 ng/ml) cell lysate at 30 µg

Lane 2:

Western blot - Human IL1B knockout THP-1 cell line (<a href='/en-us/products/cell-lines/human-il1b-knockout-thp-1-cell-line-ab273762'>ab273762</a>)

Lane 3:

IL1B knockout THP-1 untreated cell lysate at 30 µg

Lane 4:

IL1B knockout THP-1 LPS-treated (3 h, 100 ng/ml) cell lysate at 30 µg

Predicted band size: 30 kDa

Observed band size: 27-32 kDa

false