Mouse PKCz (Protein Kinase C Zeta) ELISA Kit (MOES01419)
- SKU:
- MOES01419
Description
Mouse PKCz (Protein Kinase C Zeta) ELISA Kit
This ELISA kit uses Sandwich-ELISA as the method. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to Mouse PKCz . Standards or samples are added to the appropriate micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Mouse PKCz and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well successively and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Mouse PKCz, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by adding Stop Solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of Mouse PKCz. The concentration of Mouse PKCz in samples can be calculated by comparing the OD of the samples to the standard curve.
| Assay type | Sandwich |
| Format | 96T |
| Assay time | 4.5h |
| Reactivity | Mouse |
| Detection Method | Colormetric |
| Detection Range | 0.16-10 ng/mL |
| Sensitivity | 0.10 ng/mL |
| Sample Volume Required Per Well | 100uL |
| Sample Type | Serum, plasma and other biological fluids |
Specificity
This kit recognizes Mouse PKCz in samples. No significant cross-reactivity or interference between Mouse PKCz and analogues was observed.
Typical Data
As the OD values of the standard curve may vary according to the conditions of the actual assay performance (e.g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.
| Concentration (ng/mL) | O.D | Average | Corrected |
|---|---|---|---|
| 10 | 2.311 2.359 | 2.335 | 2.246 |
| 5 | 1.665 1.715 | 1.69 | 1.601 |
| 2.5 | 0.931 0.897 | 0.914 | 0.825 |
| 1.25 | 0.509 0.543 | 0.526 | 0.437 |
| 0.63 | 0.279 0.267 | 0.273 | 0.184 |
| 0.32 | 0.192 0.188 | 0.19 | 0.101 |
| 0.16 | 0.137 0.145 | 0.141 | 0.052 |
| 0 | 0.085 0.093 | 0.089 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Mouse PKCz were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Mouse PKCz were tested on 3 different plates, 20 replicates in each plate.
| Intra-assay Precision | Inter-assay Precision | |||||
|---|---|---|---|---|---|---|
| Sample | 1 | 2 | 3 | 1 | 2 | 3 |
| n | 20 | 20 | 20 | 20 | 20 | 20 |
| Mean (ng/mL) | 0.46 | 1.55 | 3.89 | 0.44 | 1.68 | 4.01 |
| Standard deviation | 0.03 | 0.07 | 0.20 | 0.02 | 0.09 | 0.19 |
| C V (%) | 6.52 | 4.52 | 5.14 | 4.55 | 5.36 | 4.74 |
Recovery
The recovery of Mouse PKCz spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.
| Sample Type | Range (%) | Average Recovery (%) |
|---|---|---|
| Serum (n=5) | 85-96 | 91 |
| EDTA plasma (n=5) | 91-103 | 97 |
| Cell culture media (n=5) | 89-103 | 96 |
Linearity
Samples were spiked with high concentrations of Mouse PKCz and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.
| Serum (n=5) | EDTA plasma (n=5) | Cell culture media (n=5) | ||
|---|---|---|---|---|
| 1:2 | Range (%) | 96-110 | 99-114 | 85-97 |
| Average (%) | 104 | 104 | 92 | |
| 1:4 | Range (%) | 89-103 | 86-101 | 84-98 |
| Average (%) | 94 | 93 | 89 | |
| 1:8 | Range (%) | 94-108 | 83-96 | 85-99 |
| Average (%) | 100 | 88 | 90 | |
| 1:16 | Range (%) | 87-101 | 84-95 | 81-93 |
| Average (%) | 93 | 88 | 87 |
Kit Components & Storage
An unopened kit can be stored at 4'C for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.
| Item | Specifications | Storage |
|---|---|---|
| Micro ELISA Plate(Dismountable) | 8 wells X12 strips | -20'C, 6 months |
| Reference Standard | 2 vials | |
| Concentrated Biotinylated Detection Ab (100X) | 1 vial, 120 uL | |
| Concentrated HRP Conjugate (100X) | 1 vial, 120 uL | -20'C(shading light), 6 months |
| Reference Standard & Sample Diluent | 1 vial, 20 mL | 4'C, 6 months |
| Biotinylated Detection Ab Diluent | 1 vial, 14 mL | |
| HRP Conjugate Diluent | 1 vial, 14 mL | |
| Concentrated Wash Buffer (25X) | 1 vial, 30 mL | |
| Substrate Reagent | 1 vial, 10 mL | 4'C(shading light) |
| Stop Solution | 1 vial, 10 mL | 4'C |
| Plate Sealer | 5 pieces | |
| Product Description | 1 copy | |
| Certificate of Analysis | 1 copy |
Mouse PKCz (Protein Kinase C Zeta) ELISA Kit (MOES01419) Assay procedure
- 1. Set standard, test sample and control (zero) wells on the pre-coated plate and record theirpositions. It is recommended to measure each standard and sample in duplicate. Note: addall solutions to the bottom of the plate wells while avoiding contact with the well walls. Ensuresolutions do not foam when adding to the wells.
- 2. Aliquot 100µl of standard solutions into the standard wells.
- 3. Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
- 4. Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids) into test sample wells.
- 5. Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
- 6. Aspirate the liquid from each well, do not wash. Immediately add 100µL of BiotinylatedDetection Ab working solution to each well. Cover the plate with a plate seal and gently mix.Incubate for 1 hour at 37°C.
- 7. Aspirate or decant the solution from the plate and add 350µL of wash buffer to each welland incubate for 1-2 minutes at room temperature. Aspirate the solution from each well andclap the plate on absorbent filter paper to dry. Repeat this process 3 times. Note: a microplatewasher can be used in this step and other wash steps.
- 8. Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
- 9. Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
- 10. Add 90µL of Substrate Reagent to each well. Cover with a new plate seal and incubate forapproximately 15 min at 37°C. Protect the plate from light. Note: the reaction time can beshortened or extended according to the actual color change, but not by more than 30min.
- 11. Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
- 12. Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.
Mouse PKCz (Protein Kinase C Zeta) ELISA Kit (MOES01419) Protein Information
| UniProt Protein Function: | PKCZ: an AGC kinase of the PKC family. An atypical PKC: its activity is not regulated by Ca2+, PS, DAG or phorbol esters. Constitutively active. |
| UniProt Protein Details: | Protein type:EC 2.7.11.13; Protein kinase, AGC; Protein kinase, Ser/Thr (non-receptor); Kinase, protein; AGC group; PKC family; Iota subfamily Cellular Component: apical cortex; apical plasma membrane; axon hillock; cell cortex; cytoplasm; cytosol; filamentous actin; intercellular junction; intracellular membrane-bound organelle; leading edge; lipid raft; microtubule organizing center; nuclear envelope; nuclear matrix; nucleus; perinuclear region of cytoplasm; plasma membrane; protein complex; tight junction; vesicle Molecular Function:ATP binding; phospholipase binding; potassium channel regulator activity; protein binding; protein domain specific binding; protein kinase activity; protein kinase binding; protein kinase C activity; protein serine/threonine kinase activity Biological Process: actin cytoskeleton reorganization; activation of NF-kappaB transcription factor; activation of protein kinase B; cell migration; cell surface receptor linked signal transduction; cellular response to insulin stimulus; establishment of cell polarity; insulin receptor signaling pathway; long-term memory; membrane depolarization; membrane hyperpolarization; microtubule cytoskeleton organization and biogenesis; negative regulation of apoptosis; negative regulation of hydrolase activity; negative regulation of insulin receptor signaling pathway; negative regulation of peptidyl-tyrosine phosphorylation; negative regulation of protein complex assembly; peptidyl-serine phosphorylation; phospholipase D activation; positive regulation of cell proliferation; positive regulation of cell-matrix adhesion; positive regulation of glucose import; positive regulation of insulin receptor signaling pathway; positive regulation of interleukin-4 production; positive regulation of protein transport; positive regulation of synaptic transmission; positive regulation of T-helper 2 cell differentiation; protein amino acid phosphorylation; protein heterooligomerization; signal transduction; vesicle transport along microtubule |
| UniProt Code: | Q02956 |
| NCBI GenInfo Identifier: | 341940889 |
| NCBI Gene ID: | 18762 |
| NCBI Accession: | Q02956.2 |
| UniProt Secondary Accession: | Q02956,A2AD76, |
| UniProt Related Accession: | Q02956 |
| Molecular Weight: | 67,682 Da |
| NCBI Full Name: | Protein kinase C zeta type |
| NCBI Synonym Full Names: | protein kinase C, zeta |
| NCBI Official Symbol: | Prkcz |
| NCBI Official Synonym Symbols: | Pkcz; C80388; R74924; zetaPKC; AI098070; aPKCzeta; nPKC-zeta |
| NCBI Protein Information: | protein kinase C zeta type |
| UniProt Protein Name: | Protein kinase C zeta type |
| UniProt Synonym Protein Names: | nPKC-zeta |
| Protein Family: | Protein kinase |
| UniProt Gene Name: | Prkcz |
| UniProt Entry Name: | KPCZ_MOUSE |
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