All lanes : Anti-REDD-1/DDIT4 antibody [EPR18716] (ab191871) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : DDIT4 knockout HeLa cell lysate
Lane 3 : HeLa treated with 0.5mM CoCL2 for 6 hours cell lysate
Lane 4 : Untreated HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

Predicted band size: 25 kDa
Observed band size: 35 kDa why is the actual band size different from the predicted?

Lanes 1-4: Merged signal (red and green). Green - ab191871 observed at 35 kDa. Red - loading control ab8245 observed at 36 kDa. 

 ab191871 Anti-REDD-1/DDIT4 antibody [EPR18716] was shown to specifically react with REDD-1/DDIT4 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab264834 (knockout cell lysate ab257409) was used.  Wild-type and REDD-1/DDIT4 knockout samples were subjected to SDS-PAGE.  ab191871 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively.  Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Immunofluorescent analysis of 100 methanol-fixed HeLa (Human epithelial cell line from cervix adenocarcinoma) cells, untreated or treated with CoCl₂ (0.5 mM) for 6 hours, labeling REDD-1/DDIT4 with ab191871 at 1/250 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on HeLa cell line. The expression increased after treatment with CoCl₂ (0.5 mM) for 6 hours. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor^®594 Goat anti-Mouse secondary) at 1/1000 dilution (red).

The negative controls are as follows:
-ve control 1: ab191871 at 1/250 dilution followed by ab150120  at 1/1000 dilution.
-ve control 2: ab7291  at 1/1000 dilution followed by ab150077  at 1/1000 dilution.

All lanes : Anti-REDD-1/DDIT4 antibody [EPR18716] (ab191871) at 1/1000 dilution

Lane 1 : Untreated HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2 : HeLa (Human epithelial cell line from cervix adenocarcinoma) treated with 0.5 mM CoCl2 for 6 hours whole cell lysate
Lane 3 : Untreated MCF7 (Human breast adenocarcinoma cell line) whole cell lysate
Lane 4 : MCF7 (Human breast adenocarcinoma cell line) treated with 0.5 mM CoCl2 for 6 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: 35 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

REDD-1/DDIT4 was immunoprecipitated from 1mg of HeLa (Human epithelial cell line from cervix adenocarcinoma) treated with 0.5mM CoCl₂ for 6 hours whole cell lysate with ab191871 at 1/50 dilution. Western blot was performed from the immunoprecipitate using ab191871 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1: HeLa treated with 0.5mM CoCl₂ for 6h whole cell lysate, 10µg (Input).

Lane 2: ab191871 IP in HeLa treated with 0.5mM CoCl₂ for 6h whole cell lysate.

Lane 3: Rabbit IgG,monoclonal- Isotype Control (ab172730) instead of ab191871 in HeLa treated with 0.5mM CoCl₂ for 6h whole cell lysate.

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

Exposure time: 1 second.