All lanes : Anti-Catalase antibody [EPR20198] (ab209211) at 1/2000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : CAT knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 60 kDa
Observed band size: 60 kDa

Lanes 1-2: Merged signal (red and green). Green - ab209211 observed at 60 kDa. Red - loading control ab8245 observed at 37 kDa.

 ab209211 Anti-Catalase antibody [EPR20198] was shown to specifically react with Catalase in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265250 (knockout cell lysate ab256859) was used. Wild-type and Catalase knockout samples were subjected to SDS-PAGE. ab209211 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 2000 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 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized C2C12 (Mouse myoblast cell line) cells labeling Catalase with ab209211 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on C2C12 cell line. The nuclear counterstain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) at 1/200 dilution (red).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution.

All lanes : Anti-Catalase antibody [EPR20198] (ab209211) at 1/2000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : CAT knockout HAP1 whole cell lysate
Lane 3 : Hek293 whole cell lysate
Lane 4 : HepG2 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 60 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab209211 observed at 60 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab209211 was shown to specifically react with CAT in wild-type HAP1 cells. No band was observed when CAT knockout samples were examined. Wild-type and CAT knockout samples were subjected to SDS-PAGE. Ab209211 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/2000 dilution and 1/10,000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) ab216776 secondary antibodies at 1/10,000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-Catalase antibody [EPR20198] (ab209211) at 1/2000 dilution

Lane 1 : Mouse brain lysate
Lane 2 : Mouse heart lysate
Lane 3 : Mouse spleen lysate
Lane 4 : Mouse kidney lysate
Lane 5 : Rat heart lysate
Lane 6 : Rat brain lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 60 kDa
Observed band size: 60 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1-3: 5 seconds; Lane 4/5: 1 second; Lane 6: 3 seconds.

All lanes : Anti-Catalase antibody [EPR20198] (ab209211) at 1/2000 dilution

Lane 1 : C6 (Rat glial tumor cell line) whole cell lysate
Lane 2 : PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate
Lane 3 : NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate
Lane 4 : HepG2 (Human liver hepatocellular carcinoma cell line) whole cell lysate
Lane 5 : HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate
Lane 6 : C2C12 (Mouse myoblast cell line) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 60 kDa
Observed band size: 60 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1: 1 second; Lane 2/3/6: 3 seconds; Lane 4/5: 30 seconds.

All lanes : Anti-Catalase antibody [EPR20198] (ab209211) at 1/2000 dilution

Lane 1 : Human fetal brain lysate
Lane 2 : Human fetal heart lysate
Lane 3 : Human fetal kidney lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 60 kDa
Observed band size: 60 kDa


Exposure time: 1 minute

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (Mouse embryonic fibroblast cell line) cells labeling Catalase with ab209211 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on NIH/3T3 cell line. The nuclear counterstain is DAPI (blue).

Tubulin is detected with  Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) at 1/200 dilution (red).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution.

Flow cytometric analysis of 4% paraformaldehyde-fixed C2C12 (Mouse myoblast cell line) cells labeling Catalase with ab209211 at 1/60 dilution (red) compared with Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730; black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat Anti-Rabbit IgG (Alexa Fluor^® 488) at 1/2000 dilution was used as the secondary antibody.