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The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 1 µg/ml. Detects a band of approximately 82 kDa (predicted molecular weight: 82 kDa).
Abcam recommends using milk as the blocking agent. Abcam welcomes customer feedback and would appreciate any comments regarding this product.
Use a concentration of 10 µg/ml.
This is a calcium-independent, phospholipid-dependent, serine- and threonine-specific enzyme. Essential for T-cell receptor (TCR)-mediated T-cell activation, but is dispensable during TCR-dependent thymocyte development. Links the TCR signaling complex to the activation of NF-kappa-B in mature T lymphocytes. Required for interleukin-2 (IL2) production. PKC is activated by diacylglycerol which in turn phosphorylates a range of cellular proteins. PKC also serves as the receptor for phorbol esters, a class of tumor promoters.
Skeletal muscle, megakaryoblastic cells and platelets.
Belongs to the protein kinase superfamily. AGC Ser/Thr protein kinase family. PKC subfamily. Contains 1 AGC-kinase C-terminal domain. Contains 1 C2 domain. Contains 2 phorbol-ester/DAG-type zinc fingers. Contains 1 protein kinase domain.
The C1 domain, containing the phorbol ester/DAG-type region 1 (C1A) and 2 (C1B), is the diacylglycerol sensor and the C2 domain is a non-calcium binding domain.
Autophosphorylation at Thr-219 is required for targeting to the TCR and cellular function of PKC upon antigen receptor ligation.
ICC/IF image of ab109481 stained HepG2 cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab109481 at 10µg/ml overnight at +4°C. The secondary antibody (pseudo-colored green) was a goat anti-rabbit Alexa Fluor® 488 (ab150081) IgG (H+L) preadsorbed, used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1h at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43µM for 1hour at room temperature.
Western blot - Anti-PKC theta antibody (ab109481)
All lanes : Anti-PKC theta antibody (ab109481) at 1 µg/ml (Milk block 3%)
Lane 1 : Human skeletal muscle tissue lysate - total protein (ab29330) Lane 2 : Skeletal Muscle (Mouse) Tissue Lysate
Lysates/proteins at 10 µg per lane.
Secondary All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution
Developed using the ECL technique.
Performed under reducing conditions.
Predicted band size: 82 kDa Observed band size: 82 kDa Additional bands at: 105 kDa. We are unsure as to the identity of these extra bands.
Exposure time: 20 minutes
This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 3% Milk before being incubated with ab109481 overnight at 4°C. Antibody binding was detected using an anti-rabbit HRP secondary antibody, and visualised using ECL development solution ab133406.