|Reactivity||Human Mouse Rat|
|Storage buffer||10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide.|
|Storage||Store at –20°C.|
1. Aspirate media from cultures and Wash the cells with 1X PBS. 2. Lyse cells by adding 1X SDS sample buffer and transfer the extract to a microcentrifuge tube. Keep onice. 3. Sonicate for 10–15 sec to complete cell lysis and shear DNA. 4. Heat a 20 µl sample to 95–100°C for 5 min, then cool on ice. 5. Centrifuge for 5 min (with Microcentrifuge). 6. Load appropriate volumes of samples onto SDS-PAGE gel (loading quantity of protein sample depends on the concentration of extracted proteins). NOTE: At the same time, please load the pre-stained molecular weight markers to determine molecular weights and verify electrotransfer. 7. Electrotransfer to nitrocellulose/PVDF membrane.
Membrane Blocking and Antibody Incubations
1. (Optional) After transfer, wash the transferred membrane with TBS for 5 min at room temperature. 2. Incubate the membrane in the blocking buffer for 1 hr at room temperature. 3. Wash three times for 5 min each with TBST.
b. Antibodies Incubation
1. Incubate membrane and primary antibody (at the appropriate dilution and diluent recommended) in a primary antibody dilution buffer with gentle agitation overnight at 4°C. 2. Wash three times for 5 min each with TBST. 3. Incubate membrane with an appropriate second antibodydissolved in the blocking buffer with gentle agitation for 1 hr at room temperature. 4. Wash three times for 5 min each with TBST. 5. Proceed with detection.
Detection of Proteins
1. After antibodies incubation, Wash membrane three times for 5 minutes in TBST. 2. PrepareECL Reagent (or other chromogenic agents/substrate according to your second antibody). Mix well. 3. Incubate substrate with membrane for 1 minute, remove excess solution (membrane remains wet), wrap in plastic and expose to X-ray film.
|Specificity||PKC theta Rabbit Recombinant mAb detects endogenous level of total PKC theta.|
|Background||PKC includes a large family of homologous serine/threonine protein kinases that is widely conserved in eukaryotes. In mammals, there are 12 isoforms that are identified and subdivided into three groups based on their divergent regulatory domains and their second messenger requirements for activation: the conventional PKCs (cPKCs: α, βI, βII, and γ); novel PKCs (nPKCs: δ, ϵ, η, and θ); and atypical PKCs (aPKCs: ι/λ) and ζ). The intrinsic PKC-θ kinase activity is regulated through an allosteric mechanism, which leads to the change in PKC-θ conformation between “closed/inactive” and “open/active” state. Upon the initial receptor stimulation, PKC-θ is recruited to plasma membrane via membrane-resident DAG binding to its C1 domain. This triggers the conformational change of PKC-θ from “close” to “open” state, which allows its activation loop in the kinase domain to be accessible for the phosphorylation by germinal center kinase-like kinase (GLK, also known as MAP4K3). It plays an integral role in activating a range of signaling cascades that ultimately results in a transcriptional network in T cells.|