Lanes 1 - 6: Merged signal (red and green). Green - ab183218 observed at 26 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55 kDa.

ab183218 was shown to react with TNF alpha in THP-1 wild-type cells in Western blot with loss of signal observed in TNF knockout sample. Wild-type and TNF knockout THP-1 cell lysates were subjected to SDS-PAGE. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with ab183218 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.

Immunocytochemistry/ Immunofluorescence analysis of RAW264.7(Mouse Abelson murine leukemia virus-induced tumor macrophage) treated with 100ng/ml LPS for 7 h and 1µg/ml BFA for the last 3h cells labeling TNF alpha with purified ab183218 at 1/5000 dilution (0.09 µg/ml). Cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1/200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1/1000 dilution (2 µg/ml) dilution. DAPI was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Blocking/Dilution buffer : 5% NFDM/TBST.

Blocking/Dilution buffer: 5% NFDM/TBST.

The 33kDa band probably is the dimeric intermediate form that has been described in the literature (PMID: 9933416).

TNF alpha was immunoprecipitated from 0.35 mg of TPA pretreated THP-1 (Human monocytic leukemia cell line) whole cell lysate treated with 100 ng/ml LPS for 4 hours, then added 1 μg/ml BFA for 3 hours with ab183218 at 1/40 dilution. Western blot was performed from the immunoprecipitate using ab183218 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.

Lane 1: TPA pretreated THP-1 whole cell lysate treated with 100 ng/ml LPS for 4 hours, then added 1 μg/ml BFA for 3 hours 10µg (Input).

Lane 2: ab183218 IP in TPA pretreated THP-1 whole cell lysate treated with 100 ng/ml LPS for 4 hours, then added 1 μg/ml BFA for 3 hours.

Lane 3: Rabbit IgG,monoclonal [EPR25A]-Isotype Control (ab172730) instead of ab183218 in TPA pretreated THP-1 whole cell lysate treated with 100 ng/ml LPS for 4 hours, then added 1 μg/ml BFA for 3 hours.

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

Exposure time: 1 second.