|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 eta Rabbit Recombinant mAb detects endogenous level of total PKC eta.|
|Background||Protein kinase C (PKC) is known as a key enzyme in signal transduction, which is involved in the regulation of numerous cellular functions. PKC isoforms comprise a family of serine/threonine kinases, the activity of which is typically initiated by lipid second messengers on the plasma membrane. The phosphorylation of PKC molecules by upstream kinases. PKC eta is predominantly expressed in squamous epithelia or epithelia from which squamous cell carcinomas arise. These include skin, tongue, esophagus, forestomach, trachea and bronchus. In primary human keratinocytes, PKCB is located in the cytoplasm particularly in the perinuclear region, but not in the nucleus. PKC eta is located on the rough endoplasmic reticulum and on the outer nuclear membrane, which is continuous with the endoplasmic reticulum membrane. PKC eta acts as a key regulator in early B-cell development. It plays an important role in the regulation of cell division and cell death during early B-cell development. PKC eta participates in various signal transduction cascades by cross talk with complex signal-transduction networks, such as the RasIMAPK pathway, tyrosine kinase pathways, PKCμ/PKD pathway, and cyclin/cdk pathways.|