All lanes : Anti-TNF alpha antibody [EPR22598-212] (ab255275) 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 - ab255275 observed at 26 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55 kDa.

ab255275 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 ab255275 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.

Flow cytometric analysis of 2% paraformaldehyde-fixed, 0.1% Tween 20 permeabilized Human peripheral blood mononuclear cell (PBMC) treated with 50ng/ml PMA, 250ng/ml ionomycin and 300ng/ml Brefeldin A for 16h, labeling TNF alpha with ab255275 at 1/700 dilution (Right) compared with Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (Left).

Goat Anti-Rabbit IgG Fc (Alexa Fluor^® 488) preadsorbed (ab150097), at 1/5000 dilution was used as the secondary antibody.

Cells were surface stained with anti-CD3 conjugated to Alexa Fluor^® 647. Then fixed with 2% PFA for 10min and intracellularly stained with rabbit IgG (Left) or ab236836 (Right).

 

All lanes : Anti-TNF alpha antibody [EPR22598-212] (ab255275) at 1/1000 dilution

Lane 1 : THP-1 (human monocytic leukemia cell line) (treated with 80nM 12-O-Tetradecanoylphorbol-13-acetate (TPA) overnight) whole cell lysate
Lane 2 : THP-1 (treated with 80nM TPA overnight, replaced the culture medium with 100 ng/ml lipopolysaccharides (LPS) for 6 hours with addition of 300ng/ml Brefeldin A (BFA) for the last 3 hours) whole cell lysate

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: 25 kDa


Exposure time: 3 minutes

Blocking and dilution buffer: 5% NFDM/TBST.

The expression profile observed is consistent with what has been described in the literature (PMID: 9657756).

 

TNF alpha was immunoprecipitated from 0.35 mg of THP-1 (human monocytic leukemia cell line) (treated with 80nM TPA overnight, replaced the culture medium with 100 ng/ml Lipopolysaccharides (LPS) for 6 hours with addition of 300ng/ml Brefeldin A (BFA) for the last 3 hours) whole cell lysate with ab255275 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab255275 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used as secondary antibody at 1/5000 dilution.

Lane 1: THP-1 (treated as above) whole cell lysate 10 μg (Input).
Lane 2: ab255275 IP in THP-1 (treated as avove) whole cell lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab255275 in THP-1 (treated as above) whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 15 seconds.

All lanes : Anti-TNF alpha antibody [EPR22598-212] (ab255275) at 1/1000 dilution

Lane 1 : Untreated RAW 264.7 (mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate
Lane 2 : RAW 264.7 (treated with 100 ng/ml lipopolysaccharide (LPS) for 6 hours with addition of 300 ng/ml Brefeldin A (BFA) for the last 3 hours) whole cell lysate

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: 25 kDa


Exposure time: 3 minutes

Blocking and dilution buffer: 5% NFDM/TBST.

The expression profile observed is consistent with what has been described in the literature (PMID: 9657756).