All lanes : Anti-LSD2 / AOF1 antibody [EPR18508] (ab193080) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : KDM1B knockout HeLa cell lysate
Lane 3 : HAP1 cell lysate
Lane 4 : HCT116 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: 92 kDa
Observed band size: 95 kDa why is the actual band size different from the predicted?

Lanes 1-4: Merged signal (red and green). Green - ab193080 observed at 95 kDa. Red - loading control ab8245 observed at 36 kDa. 

 ab193080 Anti-LSD2 / AOF1 antibody [EPR18508] was shown to specifically react with LSD2 / AOF1 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265969 (knockout cell lysate ab258016) was used.  Wild-type and LSD2 / AOF1 knockout samples were subjected to SDS-PAGE.  ab193080 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at room temperature for 2. 5 hours 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.

Flow cytometry analysis of HeLa (human cervix adenocarcinoma) cells labelling LSD2 / AOF1 antibody (red) with purified ab193080 at a dilution of 1/70. Goat anti rabbit IgG (Alexa Fluor® 488) was used as the secondary antibody at 1/2000. Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. Isotype control antibody was Rabbit monoclonal IgG (black). The blue line shows cells without incubation with primary antibody and secondary antibody.

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: LSD2 / AOF1 knockout HAP1 cell lysate (20 µg)
Lane 3: PC12 cell lysate (20 µg)
Lane 4: Raw264.7 cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab193080 observed at 95 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab193080 was shown to specifically react with LSD2 / AOF1 when LSD2 / AOF1 knockout samples were used. Wild-type and LSD2 / AOF1 knockout samples were subjected to SDS-PAGE. ab193080 and ab8245 (loading control to GAPDH) were diluted at 1/2000 and 1/10 000 respectively and incubated overnight at 4°C. 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 1/10 000 dilution for 1 h at room temperature before imaging.

All lanes : Anti-LSD2 / AOF1 antibody [EPR18508] (ab193080) at 1/2000 dilution

Lane 1 : Human fetal brain lysate
Lane 2 : Human fetal heart lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 92 kDa
Observed band size: 92 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-LSD2 / AOF1 antibody [EPR18508] (ab193080) at 1/2000 dilution

Lane 1 : NIH/3T3 (Mouse embyro fibroblast cells) cell lysate
Lane 2 : Mouse thymus lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

Predicted band size: 92 kDa
Observed band size: 92 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-LSD2 / AOF1 antibody [EPR18508] (ab193080) at 1/2000 dilution

Lane 1 : C6 (Rat glial tumor cells) cell lysate
Lane 2 : RAW 264.7 (Mouse macrophage cells transformed with Abelson murine leukemia virus) cell lysate
Lane 3 : PC-12 (Rat adrenal gland pheochromocytoma) cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/1000 dilution

Predicted band size: 92 kDa
Observed band size: 92 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling LSD2 / AOF1 with ab193080 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing nuclear staining on HeLa cell line.

The nuclear counterstain 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: ab193080 at 1/500 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution.
-ve control 2: ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/1000 dilution.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized A431 (Human epidermoid carcinoma) cells labeling LSD2 / AOF1 with ab193080 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing nuclear staining on A431 cell line.

The nuclear counterstain 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: ab193080 at 1/500 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution.
-ve control 2: ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/1000 dilution.

LSD2 / AOF1 was immunoprecipitated from 1mg of K562 (Human chronic myelogenous leukemia cells from bone marrow) whole cell lysate with ab193080 at 1/80 dilution.

Lane 1: K562 whole cell lysate 10ug (Input).

Lane 2: ab193080 IP in K562 whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab193080 in K562 whole cell lysate.

Western blot was performed from the immunoprecipitate using ab193080 at 1/1000 dilution. Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG, was used as secondary antibody at 1/1500.

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

Exposure time: 8 seconds.