Immunofluorescent analysis of CASK was performed using 70% confluent log phase U-87 MG cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with CASK Rabbit Polyclonal Antibody (PA1-544) 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||Human, Mouse, Rat|
|Host / Isotype||Rabbit / IgG|
|Immunogen||Synthetic Peptide: C(578) E R D S P S T S R Q S P A N(592)|
|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 (IHC)||Assay dependent|
|Western Blot (WB)||0.5-2 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.
PA1-544 detects calmodulin sensitive kinase (CASK) from mouse and rat tissues and cells.
PA1-544 has been successfully used in Western blot and immunohistochemistry procedures. By Western blot, this antibody detects a 112 kDa protein representing CASK from AtT20 cell lysate. Immunohistochemical staining of CASK in mouse brain with PA1-544 results in staining of the choroid plexus.
PA1-544 immunizing peptide corresponds to amino acid residues 578-592 from human CASK. This sequence is completely conserved between rat, mouse, and human CASK.
Calmodulin sensitive kinase (CASK/mLIN-2) is a ~112-kDa member of the membrane associated quanylate kinase (MAGUK) protein family with multiple protein binding domains. These include an N-terminal calmodulin kinase (CaMKII) domain and C-terminal PDZ, SH3-, and quanylate kinase domains. CASK possesses both tyrosine kinase and serine/threonine kinase activity. In addition, CASK can form a heterotrimeric complex with Mint (X11or mLIN-10) and Veli (mLIN-7) proteins which is predicted to participate in plasma membrane receptor localization. CASK is localized to neuronal synapses and also interacts with syndecan and neurexin. The guanylate kinase domain of CASK has been shown to interact with the T-box transcription factor, Tbr-1, as a coactivator to induce transcription of genes that contain T-elements.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.