Mouse Protein kinase C epsilon type (Prkce) ELISA Kit (MOEB2305)

Product Name:	Mouse Protein kinase C epsilon type (Prkce) ELISA Kit
SKU:	MOEB2305
Size:	96T
Target:	Mouse Protein kinase C epsilon type (Prkce)
Synonyms:	nPKC-epsilon, Pkce, Pkcea
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Mouse

Intra CV:

0.0%

Inter CV:

0.0%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	104-114%	96-106%	86-98%	104-114%
EDTA Plasma(N=5)	97-106%	89-97%	109-117%	94-106%
Heparin Plasma(N=5)	89-99%	105-116%	82-92%	104-113%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	80	80-80
Plasma	80	80-80

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. 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). 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:	P16054
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant mouse 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, DGKQ and STAT3 (By similarity). Interacts with YWHAB. Interacts with HSP90AB1; promotes functional activation in a heat shock-dependent manner.
Research Area:	Cancer
Subcellular Location:	Cytoplasm Cytoplasm Cytoskeleton Cell membrane Cytoplasm Perinuclear region Nucleus Translocated to plasma membrane in epithelial cells stimulated by HGF (By similarity). Associated with the Golgi at the perinuclear site in pre-passage fibroblasts. In passaging cells, translocated to the cell periphery. Translocated to the nucleus in PMA-treated cells.
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:EC 2.7.11.13; Kinase, protein; Protein kinase, Ser/Thr (non-receptor); Protein kinase, AGC; AGC group; PKC family; Eta subfamily Cellular Component: cytoplasm; cytosol; endoplasmic reticulum; Golgi apparatus; Golgi membrane; membrane; mitochondrion; nucleus; perinuclear region of cytoplasm; plasma membrane Molecular Function:actin monomer binding; calcium-independent protein kinase C activity; enzyme activator activity; enzyme binding; ethanol binding; protein binding; protein kinase binding; protein kinase C activity; protein serine/threonine kinase activity; receptor activator activity; receptor binding; SH3 domain binding Biological Process: chemosensory behavior; lipopolysaccharide-mediated signaling pathway; macrophage activation during immune response; negative regulation of apoptosis; negative regulation of protein ubiquitination; negative regulation of release of sequestered calcium ion into cytosol; peptidyl-serine phosphorylation; positive regulation of actin filament polymerization; positive regulation of cytokinesis; positive regulation of I-kappaB kinase/NF-kappaB cascade; positive regulation of insulin secretion; positive regulation of lipid catabolic process; positive regulation of MAPKKK cascade; positive regulation of synaptic transmission, GABAergic; protein amino acid phosphorylation; reduction of mitochondrial calcium ion concentration; regulation of lipid metabolic process; regulation of peptidyl-tyrosine phosphorylation; regulation of release of sequestered calcium ion into cytosol; release of sequestered calcium ion into cytosol; response to morphine
UniProt Code:	P16054
NCBI GenInfo Identifier:	125555
NCBI Gene ID:	18754
NCBI Accession:	P16054.1
UniProt Related Accession:	P16054
Molecular Weight:	83,561 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; PKC[e]; R75156; PKCepsilon; 5830406C15Rik
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_MOUSE

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.