重组Anti-Creatine kinase B type抗体[EPR3927] (ab92452)

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.

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.

Intracellular 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).

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 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.