All lanes : Anti-Casein Kinase 1 delta/CSNK1D antibody [AF12G4] (ab85320) at 1/500 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : CSNK1D knockout HeLa cell lysate
Lane 3 : K-562 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 47 kDa
Observed band size: 47 kDa

Lanes 1-3: Merged signal (red and green). Green - ab85320 observed at 47 kDa. Red - loading control, ab181602 observed at 37 kDa.

ab85320 Anti-Casein Kinase 1 delta/CSNK1D antibody [AF12G4] was shown to specifically react with Casein Kinase 1 delta/CSNK1D in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab264667 (knockout cell lysate ab258383) was used. Wild-type and Casein Kinase 1 delta/CSNK1D knockout samples were subjected to SDS-PAGE. ab85320 and Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) were incubated overnight at 4°C at 1 in 500 dilution and 1 in 20000 dilution respectively. 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 in 10000 dilution for 1 hour at room temperature before imaging.

 

All lanes : Anti-Casein Kinase 1 delta/CSNK1D antibody [AF12G4] (ab85320) at 1/5000 dilution

Lane 1 : HeLa cell lysate
Lane 2 : K562 cell lysate
Lane 3 : SK-N-MC cell lysate
Lane 4 : 293T cell lysate
Lane 5 : L929 cell lysate

Predicted band size: 47 kDa
Observed band size: 47 kDa

Overlay histogram showing HeLa cells stained with ab85320 (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 (ab85320, 1µg/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 IgG2b [PLPV219] (ab91366, 2µg/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.

IHC image of ab85320 staining in human Alzheimer brain (hippocampus) formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab85320, 0.2µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

Casein Kinase 1 delta/CSNK1D was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Mouse monoclonal to Casein Kinase 1 delta / CSNK1D and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, Hela whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab85320.
Secondary: Goat polyclonal to mouse IgG light chain specific (HRP) at 1/5000 dilution.
Band: 47kDa: Casein Kinase 1 delta/CSNK1D; non specific - 40kDa: We are unsure as to the identity of this extra band.