Rat PKB (Protein Kinase B) ELISA Kit (RTES00674)
- SKU:
- RTES00674
- Product Type:
- ELISA Kit
- Size:
- 96 Assays
- Uniprot:
- P47196
- Sensitivity:
- 1.88IU/mL
- Range:
- 3.13-200IU/mL
- ELISA Type:
- Sandwich
- Reactivity:
- Rat
- Tested Sample Types:
- Serum, plasma and other biological fluids
Description
| Assay type: | Sandwich |
| Format: | 96T |
| Assay time: | 4.5h |
| Reactivity: | Rat |
| Detection Method: | Colormetric |
| Detection Range: | 3.13-200 IU/mL |
| Sensitivity: | 1.88 IU/mL |
| Sample Volume Required Per Well: | 100µL |
| Sample Type: | Serum, plasma and other biological fluids |
| Specificity: | This kit recognizes Rat PKB in samples. No significant cross-reactivity or interference between Rat PKB and analogues was observed. |
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 Rat PKB. 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 Rat PKB 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 Rat PKB, 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 Rat PKB. The concentration of Rat PKB in samples can be calculated by comparing the OD of the samples to the standard curve.
| UniProt Protein Function: | Akt1: an oncogenic AGC kinase that plays a critical role in regulating cell survival and metabolism in many different signaling pathways. Dual phosphorylation is required for its activation. T308 is phosphorylated by PDK1 in the PI3 kinase pathway, and S473 is phosphorylated by mTOR in the mTORC2 pathway. The 'Lys-63'-linked ubiquitination of AKT1 by TRAF6 is important for its translocation to the plasma membrane, phosphorylation, and activation. When Akt is fully phosphorylated it translocates into the nucleus, undergoes 'Lys-48'-polyubiquitination catalyzed by TTC3, leading to its proteosomal degradation. Hyperactive or overexpressed in a number of cancers including breast, prostate, lung, pancreatic, liver, ovarian and colorectal. Over 160 protein substrates are known including many that regulate transcription, metabolism, apoptosis, cell cycle, and growth. |
| UniProt Protein Details: | Protein type:EC 2. 7. 11. 1; Protein kinase, Ser/Thr (non-receptor); Protein kinase, AGC; Oncoprotein; Kinase, protein; AGC group; AKT family Chromosomal Location of Human Ortholog: 14q32. 32 Cellular Component: nucleoplasm; microtubule cytoskeleton; mitochondrion; cytoplasm; plasma membrane; spindle; intercellular junction; nucleus; cytosol Molecular Function:identical protein binding; protein serine/threonine kinase activity; protein binding; phosphatidylinositol-3,4,5-triphosphate binding; enzyme binding; protein kinase C binding; nitric-oxide synthase regulator activity; protein serine/threonine/tyrosine kinase activity; kinase activity; phosphatidylinositol-3,4-bisphosphate binding; ATP binding; protein kinase activity Biological Process: negative regulation of JNK cascade; positive regulation of nitric oxide biosynthetic process; regulation of myelination; nerve growth factor receptor signaling pathway; protein ubiquitination; glucose homeostasis; regulation of cell migration; protein amino acid phosphorylation; G1/S-specific positive regulation of cyclin-dependent protein kinase activity; germ cell development; positive regulation of glucose import; cell projection organization and biogenesis; protein catabolic process; maternal placenta development; response to food; platelet activation; glycogen biosynthetic process; fibroblast growth factor receptor signaling pathway; positive regulation of nitric-oxide synthase activity; positive regulation of blood vessel endothelial cell migration; glucose metabolic process; positive regulation of lipid biosynthetic process; positive regulation of cell growth; insulin-like growth factor receptor signaling pathway; cellular response to insulin stimulus; response to heat; T cell costimulation; positive regulation of fat cell differentiation; negative regulation of protein kinase activity; striated muscle cell differentiation; positive regulation of transcription from RNA polymerase II promoter; positive regulation of endothelial cell proliferation; positive regulation of transcription factor activity; response to oxidative stress; regulation of nitric-oxide synthase activity; negative regulation of apoptosis; negative regulation of autophagy; negative regulation of fatty acid beta-oxidation; translation; apoptosis; protein amino acid autophosphorylation; regulation of glycogen biosynthetic process; positive regulation of cellular protein metabolic process; positive regulation of glycogen biosynthetic process; negative regulation of cell size; negative regulation of caspase activity; glucose transport; signal transduction; nitric oxide metabolic process; regulation of translation; apoptotic mitochondrial changes; protein kinase B signaling cascade; inflammatory response; nitric oxide biosynthetic process; cell differentiation; activated T cell apoptosis; aging; negative regulation of proteolysis; epidermal growth factor receptor signaling pathway; phosphoinositide-mediated signaling; myelin maintenance in the peripheral nervous system; protein modification process; endocrine pancreas development; positive regulation of peptidyl-serine phosphorylation; osteoblast differentiation; cell proliferation; G-protein coupled receptor protein signaling pathway; peptidyl-serine phosphorylation; protein import into nucleus, translocation; positive regulation of proteasomal ubiquitin-dependent protein catabolic process; insulin receptor signaling pathway; positive regulation of vasoconstriction; innate immune response; gene expression; positive regulation of protein amino acid phosphorylation; blood coagulation; vascular endothelial growth factor receptor signaling pathway; phosphorylation; hyaluronan metabolic process Disease: Schizophrenia; Cowden Syndrome 6; Proteus Syndrome; Breast Cancer; Ovarian Cancer |
| NCBI Summary: | The serine-threonine protein kinase encoded by the AKT1 gene is catalytically inactive in serum-starved primary and immortalized fibroblasts. AKT1 and the related AKT2 are activated by platelet-derived growth factor. The activation is rapid and specific, and it is abrogated by mutations in the pleckstrin homology domain of AKT1. It was shown that the activation occurs through phosphatidylinositol 3-kinase. In the developing nervous system AKT is a critical mediator of growth factor-induced neuronal survival. Survival factors can suppress apoptosis in a transcription-independent manner by activating the serine/threonine kinase AKT1, which then phosphorylates and inactivates components of the apoptotic machinery. Mutations in this gene have been associated with the Proteus syndrome. Multiple alternatively spliced transcript variants have been found for this gene. [provided by RefSeq, Jul 2011] |
| UniProt Code: | P47196 |
| NCBI GenInfo Identifier: | 62241013 |
| NCBI Gene ID: | 207 |
| NCBI Accession: | NP_001014431. 1 |
| UniProt Secondary Accession: | P47196,P31751, Q9Y243, P31750, Q60823, Q9WUA6, P47196 P47197, Q63484, |
| UniProt Related Accession: | P31749 |
| Molecular Weight: | 56kDa |
| NCBI Full Name: | RAC-alpha serine/threonine-protein kinase |
| NCBI Synonym Full Names: | AKT serine/threonine kinase 1 |
| NCBI Official Symbol: | AKT1 |
| NCBI Official Synonym Symbols: | AKT; PKB; RAC; CWS6; PRKBA; PKB-ALPHA; RAC-ALPHA |
| NCBI Protein Information: | RAC-alpha serine/threonine-protein kinase |
| UniProt Protein Name: | RAC-alpha serine/threonine-protein kinase |
| UniProt Synonym Protein Names: | Protein kinase B; PKB; Protein kinase B alpha; PKB alpha; Proto-oncogene c-Akt; RAC-PK-alpha |
| Protein Family: | AKT-interacting protein |
| UniProt Gene Name: | AKT1 |
| UniProt Entry Name: | AKT1_HUMAN |
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 (IU/mL) | O.D | Average | Corrected |
| 200 | 2.315 2.355 | 2.335 | 2.244 |
| 100 | 1.467 1.491 | 1.479 | 1.388 |
| 50 | 0.834 0.822 | 0.828 | 0.737 |
| 25 | 0.4 0.404 | 0.402 | 0.311 |
| 12.5 | 0.282 0.268 | 0.275 | 0.184 |
| 6.25 | 0.182 0.176 | 0.179 | 0.088 |
| 3.13 | 0.129 0.145 | 0.137 | 0.046 |
| 0 | 0.09 0.092 | 0.091 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Rat PKB were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Rat PKB 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 (IU/mL) | 9.89 | 22.09 | 99.27 | 9.16 | 20.79 | 104.06 |
| Standard deviation | 0.53 | 1.09 | 5.40 | 0.49 | 1.07 | 5.71 |
| C V (%) | 5.36 | 4.93 | 5.44 | 5.35 | 5.15 | 5.49 |
Recovery
The recovery of Rat PKB 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) | 88-103 | 94 |
| EDTA plasma (n=5) | 86-102 | 93 |
| Cell culture media (n=5) | 93-108 | 100 |
Linearity
Samples were spiked with high concentrations of Rat PKB 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 (%) | 91-109 | 92-104 | 96-109 |
| Average (%) | 99 | 99 | 102 | |
| 1:4 | Range (%) | 91-106 | 79-92 | 83-97 |
| Average (%) | 97 | 85 | 89 | |
| 1:8 | Range (%) | 89-104 | 82-93 | 87-100 |
| Average (%) | 95 | 87 | 93 | |
| 1:16 | Range (%) | 86-100 | 87-99 | 86-100 |
| Average (%) | 93 | 92 | 93 |
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 ×12 strips | -20°C, 6 months |
| Reference Standard | 2 vials | |
| Concentrated Biotinylated Detection Ab (100×) | 1 vial, 120 µL | |
| Concentrated HRP Conjugate (100×) | 1 vial, 120 µL | -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 (25×) | 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 |
- 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.
- Aliquot 100µl of standard solutions into the standard wells.
- Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
- Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids) into test sample wells.
- Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
- 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.
- 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.
- Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
- Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
- 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.
- Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
- Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.
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