All lanes : Anti-TNF alpha antibody [EPR19147] (ab183218) at 1/1000 dilution

Lane 1 : Wild-type THP-1 Brefeldin A (ab120299) treated (5 µg/ml, 4 h) cell lysate
Lane 2 : Wild-type THP-1 LPS treated (100 ng/ml, 16 h) and Brefeldin A (ab120299) treated (5 µg/ml, 4 h) cell lysate
Lane 3 : TNF alpha knockout THP-1 Brefeldin A (ab120299) treated (5 µg/ml, 4 h) cell lysate
Lane 4 : TNF alpha knockout THP-1 LPS treated (100 ng/ml, 16 h) and Brefeldin A (ab120299) treated (5 µg/ml, 4 h) cell lysate
Lane 5 : U937 PMA treated (10 mM, 2 days) plus 16 h no treatment and Brefeldin A (ab120299) treated (5 µg/ml, 4 h) cell lysate
Lane 6 : U937 PMA treated (10 mM, 2 days) and LPS treated (1 µg/ml, 16 h) plus Brefeldin A (ab120299) treated (5 µg/ml, 4 h) cell lysate

Lysates/proteins at 30 µg per lane.

Performed under reducing conditions.

Predicted band size: 25 kDa
Observed band size: 26 kDa why is the actual band size different from the predicted?

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.

ELISA using ab183218 between 0.1 and 1000 ng/ml to detect TNF alpha peptide. Secondary used was Alkaline Phosphatase AffiniPure Goat Anti-Rabbit IgG (H+L) 1/2000.

All lanes : Anti-TNF alpha antibody [EPR19147] (ab183218) at 1/2000 dilution

Lane 1 : TPA pretreated THP-1 (Human monocytic leukemia cell line) whole cell lysate
Lane 2 : TPA pretreated THP-1 (Human monocytic leukemia cell line) whole cell lysate treated with 100 ng/ml LPS for 7 hours
Lane 3 : 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

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 25 kDa
Observed band size: 14,26 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

The 14kDa band probably is the degradable inactive fragment that has been decribed by literature (PMID: 9933416).

All lanes : Anti-TNF alpha antibody [EPR19147] (ab183218) at 1/1000 dilution

Lane 1 : TPA pretreated RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate
Lane 2 : TPA pretreated RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate treated with 100 ng/ml LPS for 7 hours
Lane 3 : TPA pretreated RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate treated with 100 ng/ml LPS for 4 hours, then added 1 µg/ml BFA for 3 hours

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 25 kDa
Observed band size: 14,26,33 kDa why is the actual band size different from the predicted?


Exposure time: 3 seconds

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.