Immunofluorescence analysis of CaM Kinase I was performed using 70% confluent log phase U-87MG cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 2% BSA for 1 hour at room temperature. The cells were labeled with CaM Kinase I Rabbit Polyclonal Antibody (PA1543) at 2ug/ml in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (A27034) a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing cytoplasmic localization. Panel e shows the no primary antibody control. The images were captured at 60X magnification.
|Tested species reactivity||Mouse, Rat|
|Host / Isotype||Rabbit / IgG|
|Immunogen||Synthetic Peptide: A(1) V E G P R W K Q A E D I R D I Y D(18)|
|Purification||Antigen affinity chromatography|
|Storage buffer||PBS with 1mg/ml BSA|
|Contains||0.05% sodium azide|
|Storage Conditions||-20° C, Avoid Freeze/Thaw Cycles|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||3-5 µg/10^6 cells|
|Immunocytochemistry (ICC)||2 µg/ml|
|Immunofluorescence (IF)||2 µg/ml|
|Immunohistochemistry (Paraffin) (IHC (P))||1:20-1:100|
|Western Blot (WB)||1-2 µg/mL|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
PA1-543 detects calcium/calmodulin-dependent kinase I (CaMKI) from rat and mouse brain extract.
PA1-543 has been successfully used in Western blot procedures. By Western blot, this antibody detects an ~43 kDa protein representing CaMKI from rat and mouse brain extract.
PA1-543 immunizing peptide corresponds to amino acid residues 1-18 from human CaMKI protein. PA1-543 immunizing peptide (Cat. # PEP-134) is available for use in neutralization and control experiments.
Calcium/calmodulin-dependent kinases (CaMKs) are a subfamily of the serine/threonine protein kinase family that includes CaMKI, CaMKII, and CaMKIV. There have been identified multiple substrates for CaMKI, including synapsin 1, synapsin 2, CREB, and CFTR, and its activation is dependent upon phosphorylation of Thr177 by CaMK kinase (CaMKK). Studies suggest that CaMKI plays an important role in retinal function and its expression is regulated by light stimulation.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.