All lanes : Anti-DDIT3 antibody [9C8] (ab11419) at 5 µg/ml

Lane 1 : Wild-type SW480 cell lysate
Lane 2 : DDIT3 knockout SW480 cell lysate
Lane 3 : Untreated HeLa cell lysate
Lane 4 : HeLa + DMSO control cell lysate
Lane 5 : HeLa + tunicamycin (20ug/mL,4 hours) cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 19 kDa
Observed band size: 26 kDa why is the actual band size different from the predicted?

This data was developed using the same antibody clone in a different buffer formulation (ab11419).

Lanes 1 - 5: Merged signal (red and green). Green - ab11419 observed at 26 kDa. Red - loading control ab52866 (Rabbit anti-alpha Tubulin antibody [EP1332Y]) observed at 55kDa.

ab11419 was shown to react with DDIT3 in wild-type SW480 cells in western blot with loss of signal observed in DDIT3 knockout sample. Wild-type and DDIT3 knockout SW480 cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab11419 and ab52866 (Rabbit anti-alpha Tubulin antibody [EP1332Y]) overnight at 4°C at 5 µg/ml and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed (ab216777) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

ab11419 staining DDIT3 in HeLa cells +/- Tunicamycin (1.5μM, 6 hours). 

The cells were fixed with 4% PFA (10min), permeabilized with 0.1% Triton-X for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab11419 at 5μg/ml and ab6046, Rabbit polyclonal to beta Tubulin - Loading Control, at 1/1000 dilution. Cells were then incubated with ab150117, Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) at 1/1000 dilution (shown in green) and ab150084, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 594) at 1/1000 dilution (shown in pseudocolor red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, and sodium azide (ab11419).

All lanes : Anti-DDIT3 antibody [9C8] (ab11419) at 5 µg/ml

Lane 1 : HeLa w/c control cell lysate at 40 µg
Lane 2 : HeLa cells treated with 2ug/ml tunicamycin for 4 hours, whole cell lysate cell lysate at 40 µg
Lane 3 : HeLa cells treated with 20ug/ml tunicamycin for 4 hours, whole cell lysate cell lysate at 40 µg
Lane 4 : HepG2 cell lysate at 20 µg
Lane 5 : NIH3T3 cell lysate at 20 µg

Performed under reducing conditions.

Predicted band size: 19 kDa
Observed band size: 27 kDa why is the actual band size different from the predicted?

This data was developed using the same antibody clone in a different buffer formulation (ab11419).

Lanes 1 - 5: Merged signal (red and green). Green - ab11419 observed at 27 kDa. Red - loading control, Rabbit anti Actin observed at 42kDa.

ab11419 was shown to react with DDIT3 in western blot. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab11419 and Rabbit anti Actin overnight at 4°C at 5 µg/ml and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed (ab216777) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

ab11419 staining DDIT3 in SKNSH cells treated with deltamethrin (ab141019), by ICC/IF. Increase of DDIT3 expression correlates with increased concentration of deltamethrin, as described in literature.
The cells were incubated at 37°C for 48 hours in media containing different concentrations of ab141019 (deltamethrin) in DMSO, fixed with 100% methanol for 5 minutes at -20°C and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab11419 (10 μg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight^® 488 anti-mouse polyclonal antibody (ab96879) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, and sodium azide (ab11419).