Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: Creatine kinase B knockout HAP1 cell lysate (20 µg)
Lane 3: HeLa cell lysate (20 µg)
Lane 4: SH-SY5Y cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab92452 observed at 45 kDa. Red - loading control, ab18058, observed at 2X kDa.

ab92452 (unpurified) was shown to specifically react with Creatine kinase B when ProteinX knockout samples were used. Wild-type and Creatine kinase B knockout samples were subjected to SDS-PAGE. ab92452 and ab18058 (loading control to vinculin) were both diluted 1/10 000 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 1/10000 dilution for 1 hour at room temperature before imaging.

Anti-Creatine kinase B type antibody [EPR3927] (ab92452) at 1/10000 dilution (Purified) + Y79 (Human retinoblastoma retinoblastoma) whole cell lysates at 15 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 43 kDa
Observed band size: 43 kDa

Lanes 1-4 : Anti-Creatine kinase B type antibody [EPR3927] (ab92452) at 1/10000 dilution (Purified)
Lane 5 : Anti-Creatine kinase B type antibody [EPR3927] (ab92452) at 1/10000 dilution

Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates
Lane 2 : Mouse cerebellum lysates
Lane 3 : Mouse brain lysates
Lane 4 : C6 (Rat glial tumor glial cell) whole cell lysates
Lane 5 : Rat brain lysates

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 43 kDa
Observed band size: 43 kDa

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human astrocytoma tissue sections labeling Creatine kinase B type with purified ab92452 at 1/1000 dilution (0.101 µg/ml). Heat mediated antigen retrieval was performed using Bond™ Epitope Retrieval Solution 2 (pH 9.0). Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Creatine kinase B type with purified ab92452 at 1:50 dilution (2.0 µg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1:200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1:1000 (2 µg/ml) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Creatine kinase B type with purified ab92452 at 1/20 dilution (5 µg/ml) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

All lanes : Anti-Creatine kinase B type antibody [EPR3927] (ab92452) at 1/10000 dilution (unpurified)

Lane 1 : SHSY-5Y cell lysate
Lane 2 : HeLa cell lysate
Lane 3 : Y-79 cell lysate
Lane 4 : SW840 cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 43 kDa

Paraffin embedded Human glioma tissue labelled with ab92452 (unpurified) at 1/100 dilution.

Immunofluorescence analysis of HeLa cells labelled with ab92452 (unpurified) at 1/100 dilution.

Overlay histogram showing HeLa cells stained with ab92452 (unpurified) (red line). The cells were fixed with 4% paraformaldehyde (10 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 (ab92452, 1/100 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.