Description
Human Protein kinase C epsilon type (PRKCE) ELISA Kit
The Human Protein Kinase C Epsilon Type (PRKCE) ELISA Kit is a powerful tool designed for the accurate quantification of PRKCE levels in human samples including serum, plasma, and cell culture supernatants. This kit provides reliable and reproducible results with high sensitivity and specificity, making it ideal for a wide range of research applications.PRKCE is a critical kinase involved in various cellular processes, including cell growth, proliferation, and differentiation. Dysregulation of PRKCE has been linked to numerous diseases such as cancer, diabetes, and neurological disorders, making it a valuable biomarker for exploring disease mechanisms and potential therapeutic targets.
With the Human PRKCE ELISA Kit, researchers can easily and accurately measure PRKCE levels in human samples, allowing for in-depth investigations into its role in disease pathogenesis and potential therapeutic interventions. Trust in this ELISA kit for robust and precise analysis of PRKCE levels in your research studies.
| Product Name: | Human Protein kinase C epsilon type (PRKCE) ELISA Kit |
| SKU: | HUEB2566 |
| Size: | 96T |
| Target: | Human Protein kinase C epsilon type (PRKCE) |
| Synonyms: | nPKC-epsilon, PKCE |
| Assay Type: | Sandwich |
| Detection Method: | ELISA |
| Reactivity: | Human |
| Detection Range: | 1.56-100ng/mL |
| Sensitivity: | 0.98ng/mL |
| Intra CV: | 4.5% | ||||||||||||||||||||
| Inter CV: | 7.9% | ||||||||||||||||||||
| Linearity: |
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| Recovery: |
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| Function: | Calcium-independent, phospholipid- and diacylglycerol (DAG)-dependent serine/threonine-protein kinase that plays essential roles in the regulation of multiple cellular processes linked to cytoskeletal proteins, such as cell adhesion, motility, migration and cell cycle, functions in neuron growth and ion channel regulation, and is involved in immune response, cancer cell invasion and regulation of apoptosis. Mediates cell adhesion to the extracellular matrix via integrin-dependent signaling, by mediating angiotensin-2-induced activation of integrin beta-1 (ITGB1) in cardiac fibroblasts. Phosphorylates MARCKS, which phosphorylates and activates PTK2/FAK, leading to the spread of cardiomyocytes. Involved in the control of the directional transport of ITGB1 in mesenchymal cells by phosphorylating vimentin (VIM), an intermediate filament (IF) protein. In epithelial cells, associates with and phosphorylates keratin-8 (KRT8), which induces targeting of desmoplakin at desmosomes and regulates cell-cell contact. Phosphorylates IQGAP1, which binds to CDC42, mediating epithelial cell-cell detachment prior to migration. In HeLa cells, contributes to hepatocyte growth factor (HGF)-induced cell migration, and in human corneal epithelial cells, plays a critical role in wound healing after activation by HGF. During cytokinesis, forms a complex with YWHAB, which is crucial for daughter cell separation, and facilitates abscission by a mechanism which may implicate the regulation of RHOA. In cardiac myocytes, regulates myofilament function and excitation coupling at the Z-lines, where it is indirectly associated with F-actin via interaction with COPB1. During endothelin-induced cardiomyocyte hypertrophy, mediates activation of PTK2/FAK, which is critical for cardiomyocyte survival and regulation of sarcomere length. Plays a role in the pathogenesis of dilated cardiomyopathy via persistent phosphorylation of troponin I (TNNI3). Involved in nerve growth factor (NFG)-induced neurite outgrowth and neuron morphological change independently of its kinase activity, by inhibition of RHOA pathway, activation of CDC42 and cytoskeletal rearrangement. May be involved in presynaptic facilitation by mediating phorbol ester-induced synaptic potentiation. Phosphorylates gamma-aminobutyric acid receptor subunit gamma-2 (GABRG2), which reduces the response of GABA receptors to ethanol and benzodiazepines and may mediate acute tolerance to the intoxicating effects of ethanol. Upon PMA treatment, phosphorylates the capsaicin- and heat-activated cation channel TRPV1, which is required for bradykinin-induced sensitization of the heat response in nociceptive neurons. Is able to form a complex with PDLIM5 and N-type calcium channel, and may enhance channel activities and potentiates fast synaptic transmission by phosphorylating the pore-forming alpha subunit CACNA1B (CaV2.2). In prostate cancer cells, interacts with and phosphorylates STAT3, which increases DNA-binding and transcriptional activity of STAT3 and seems to be essential for prostate cancer cell invasion. Downstream of TLR4, plays an important role in the lipopolysaccharide (LPS)-induced immune response by phosphorylating and activating TICAM2/TRAM, which in turn activates the transcription factor IRF3 and subsequent cytokines production. In differentiating erythroid progenitors, is regulated by EPO and controls the protection against the TNFSF10/TRAIL-mediated apoptosis, via BCL2. May be involved in the regulation of the insulin-induced phosphorylation and activation of AKT1. |
| Uniprot: | Q02156 |
| Sample Type: | Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids |
| Specificity: | Natural and recombinant human Protein kinase C epsilon type |
| Sub Unit: | Forms a ternary complex with TRIM63 and GN2BL1. Can form a complex with PDLIM5 and N-type calcium channel. Interacts with COPB1 and YWHAB (By similarity). Interacts with DGKQ and STAT3. Interacts with HSP90AB1; promotes functional activation in a heat shock-dependent manner (PubMed:20353823). |
| Research Area: | Cancer |
| Subcellular Location: | Cytoplasm Cytoplasm Cytoskeleton Cell membrane Cytoplasm Perinuclear region Nucleus Translocated to plasma membrane in epithelial cells stimulated by HGF. Associated with the Golgi at the perinuclear site in pre-passage fibroblasts (By similarity). In passaging cells, translocated to the cell periphery (By similarity). Translocated to the nucleus in PMA-treated cells (By similarity). |
| Storage: | Please see kit components below for exact storage details |
| Note: | For research use only |
| UniProt Protein Function: | PKCE: an AGC kinase of the PKC family. Calcium-independent, phospholipid-dependent. Activated by inflammatory mediators and involved in nociceptive functions. PKC-epsilon null mice display decreased hypoxic pulmonary vasoconstriction. |
| UniProt Protein Details: | Protein type:Kinase, protein; Protein kinase, AGC; EC 2.7.11.13; Protein kinase, Ser/Thr (non-receptor); AGC group; PKC family; Eta subfamily Chromosomal Location of Human Ortholog: 2p21 Cellular Component: cytoplasm; cytosol; endoplasmic reticulum; Golgi apparatus; perinuclear region of cytoplasm; plasma membrane Molecular Function:actin monomer binding; enzyme activator activity; enzyme binding; protein binding; protein kinase C activity; protein serine/threonine kinase activity; signal transducer activity Biological Process: apoptosis; lipopolysaccharide-mediated signaling pathway; peptidyl-serine phosphorylation; phospholipase C activation; platelet activation; positive regulation of actin filament polymerization; positive regulation of cytokinesis; protein amino acid phosphorylation; signal transduction |
| NCBI Summary: | Protein kinase C (PKC) is a family of serine- and threonine-specific protein kinases that can be activated by calcium and the second messenger diacylglycerol. PKC family members phosphorylate a wide variety of protein targets and are known to be involved in diverse cellular signaling pathways. PKC family members also serve as major receptors for phorbol esters, a class of tumor promoters. Each member of the PKC family has a specific expression profile and is believed to play a distinct role in cells. The protein encoded by this gene is one of the PKC family members. This kinase has been shown to be involved in many different cellular functions, such as neuron channel activation, apoptosis, cardioprotection from ischemia, heat shock response, as well as insulin exocytosis. Knockout studies in mice suggest that this kinase is important for lipopolysaccharide (LPS)-mediated signaling in activated macrophages and may also play a role in controlling anxiety-like behavior. [provided by RefSeq, Jul 2008] |
| UniProt Code: | Q02156 |
| NCBI GenInfo Identifier: | 400135 |
| NCBI Gene ID: | 5581 |
| NCBI Accession: | Q02156.1 |
| UniProt Secondary Accession: | Q02156,Q32MQ3, Q53SL4, Q53SM5, Q9UE81, B0LPH7, |
| UniProt Related Accession: | Q02156 |
| Molecular Weight: | 83,674 Da |
| NCBI Full Name: | Protein kinase C epsilon type |
| NCBI Synonym Full Names: | protein kinase C epsilon |
| NCBI Official Symbol: | PRKCE |
| NCBI Official Synonym Symbols: | PKCE; nPKC-epsilon |
| NCBI Protein Information: | protein kinase C epsilon type |
| UniProt Protein Name: | Protein kinase C epsilon type |
| UniProt Synonym Protein Names: | nPKC-epsilon |
| Protein Family: | Protein kinase |
| UniProt Gene Name: | PRKCE |
| UniProt Entry Name: | KPCE_HUMAN |
| Component | Quantity (96 Assays) | Storage |
| ELISA Microplate (Dismountable) | 8×12 strips | -20°C |
| Lyophilized Standard | 2 | -20°C |
| Sample Diluent | 20ml | -20°C |
| Assay Diluent A | 10mL | -20°C |
| Assay Diluent B | 10mL | -20°C |
| Detection Reagent A | 120µL | -20°C |
| Detection Reagent B | 120µL | -20°C |
| Wash Buffer | 30mL | 4°C |
| Substrate | 10mL | 4°C |
| Stop Solution | 10mL | 4°C |
| Plate Sealer | 5 | - |
Other materials and equipment required:
- Microplate reader with 450 nm wavelength filter
- Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
- Incubator
- Deionized or distilled water
- Absorbent paper
- Buffer resevoir
*Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.
| Step | |
| 1. | Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C. |
| 2. | Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform. |
| 3. | Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper. |
| 4. | Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C. |
| 5. | Repeat the wash process for five times as conducted in step 3. |
| 6. | Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction. |
| 7. | Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing. |
| 8. | Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters. |
| 9. | After experiment, store all reagents according to the specified storage temperature respectively until their expiry. |
When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.
| Sample Type | Protocol |
| Serum | If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. |
| Plasma | Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit. |
| Urine & Cerebrospinal Fluid | Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid. |
| Cell culture supernatant | Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately. |
| Cell lysates | Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C. |
| Tissue homogenates | The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C. |
| Tissue lysates | Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C. |
| Breast Milk | Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles. |
