The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 0.2 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
Use 1µg for 106 cells. ab170192-Mouse monoclonal IgG2b, is suitable for use as an isotype control with this antibody.
1/5000. Detects a band of approximately 47 kDa (predicted molecular weight: 47 kDa).
Use at an assay dependent concentration.
Casein kinases are operationally defined by their preferential utilization of acidic proteins such as caseins as substrates. It can phosphorylate a large number of proteins. Participates in Wnt signaling. Central component of the circadian clock. May act as a negative regulator of circadian rhythmicity by phosphorylating PER1 and PER2. Retains PER1 in the cytoplasm.
Expressed in all tissues examined, including brain, heart, lung, liver, pancreas, kidney, placenta and skeletal muscle. In blood, highly expressed in hemopoietic cells and mature granulocytes. Also found in monocytes and lymphocytes.
Involvement in disease
Defects in CSNK1D are a cause of familial advanced sleep-phase syndrome (FASPS) [MIM:604348]. FASPS is characterized by very early sleep onset and offset. Individuals are 'morning larks' with a 4 hours advance of the sleep, temperature and melatonin rhythms.
Belongs to the protein kinase superfamily. CK1 Ser/Thr protein kinase family. Casein kinase I subfamily. Contains 1 protein kinase domain.
Autophosphorylated on serine and threonine residues.
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 BondTM 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 was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Mouse monoclonal to Casein Kinase 1 delta 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 70oC; 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; non specific - 40kDa: We are unsure as to the identity of this extra band.