All lanes : Anti-TNFAIP3 antibody [59A426] (ab13597) at 1/500 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : TNFAIP3 knockout HeLa cell lysate
Lane 3 : Jurkat cell treated with 5ng/ml PMA for 48 hours and then treated with 2µg/ml PHA for 48 hours, whole cell lysate
Lane 4 : Untreated Jurkat cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Observed band size: 80 kDa why is the actual band size different from the predicted?

Lanes 1-4: Merged signal (red and green). Green - ab13597 observed at 80 kDa. Red - loading control, ab181602 observed at 37 kDa.

ab13597 Anti-TNFAIP3 antibody [59A426] was shown to specifically react with TNFAIP3 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265983 (knockout cell lysate ab257112) was used. Wild-type and TNFAIP3 knockout samples were subjected to SDS-PAGE. ab13597 and Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) were incubated overnight at 4°C at 1 in 500 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preadsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preadsorbed (ab216777) secondary antibodies at 1 in 10000 dilution for 1 hour at room temperature before imaging.

 

Anti-TNFAIP3 antibody [59A426] (ab13597) at 4 µg/ml + Jurkat cell lysate

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human placenta tissue labelling TNFAIP3 with ab13597 at 5µg/ml. Staining was enhanced by boiling tissue sections in 10mM sodium citrate buffer, pH6.0 for 10-20 minutes followed by cooling at room temperature for 20 minutes.

All lanes : Anti-TNFAIP3 antibody [59A426] (ab13597) at 1 µg/ml

Lane 1 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate
Lane 2 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Observed band size: 90 kDa why is the actual band size different from the predicted?
Additional bands at: 15 kDa, 34 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 20 minutes

Overlay histogram showing HepG2 cells stained with ab13597 (red line). The cells were fixed with methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab13597, 2µg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a decreased signal in HepG2 cells fixed with methanol (5 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.