Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: CSNK2A1 knockout HAP1 cell lysate (20 µg)
Lane 3: HeLa cell lysate (20 µg)
Lane 4: Jurkat cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab70774 observed at 44 kDa. Red - loading control, ab181602, observed at 37 kDa.

ab70774 was shown to recognize CSNK2A1 when CSNK2A1 knockout samples were used, along with additional cross-reactive bands. Wild-type and CSNK2A1 knockout samples were subjected to SDS-PAGE. ab70774 and ab181602 (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-Mouse IgG H&L (IRDye^® 800CW) preadsorbed (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye^® 680RD) preadsorbed (ab216777) secondary antibodies at 1/10 000 dilution for 1 h at room temperature before imaging.

ab70774 (4µg/ml) staining CSNK2A1 in human cerebellum, using an automated system (DAKO Autostainer Plus). Using this protocol there is nuclear and cytoplasmic staining of purkinje cells.
Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer EDTA pH 9.0 in a DAKO PT link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako envision flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that, for manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.

All lanes : Anti-CSNK2A1 antibody [8E5] (ab70774) at 1/2000 dilution

Lane 1 : Jurkat T cell lysate
Lane 2 : K562 cell lysate
Lane 3 : NIH3T3 cell lysate
Lane 4 : C6 cell lysate

Predicted band size: 45 kDa

Overlay histogram showing Jurkat cells stained with ab70774 (red line). The cells were fixed with 80% methanol (5 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 (ab70774, 0.5µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG2a [ICIGG2A] (ab91361, 1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in Hela cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.