Mouse PKB (Protein Kinase B) ELISA Kit (MOES01412)
- Product Type:
- ELISA Kit
- 96 Assays
- ELISA Type:
- Tested Sample Types:
- Serum, plasma and other biological fluids
|Detection Range:||0.78-50 ng/mL|
|Sample Volume Required Per Well:||100µL|
|Sample Type:||Serum, plasma and other biological fluids|
|Specificity:||This kit recognizes Mouse PKB in samples. No significant cross-reactivity or interference between Mouse 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 Mouse 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 Mouse 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 Mouse 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 Mouse PKB. The concentration of Mouse 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; Oncoprotein; Kinase, protein; Protein kinase, AGC; Protein kinase, Ser/Thr (non-receptor); AGC group; AKT family
Cellular Component: cytoplasm; cytosol; intercellular junction; microtubule cytoskeleton; mitochondrion; nucleus; plasma membrane; protein complex; spindle; vesicle
Molecular Function:ATP binding; enzyme binding; GTPase activating protein binding; identical protein binding; kinase activity; nitric-oxide synthase regulator activity; phosphatidylinositol-3,4,5-triphosphate binding; phosphatidylinositol-3,4-bisphosphate binding; protein binding; protein kinase activity; protein kinase binding; protein kinase C binding; protein phosphatase 2A binding; protein serine/threonine kinase activity; protein serine/threonine/tyrosine kinase activity
Biological Process: activated T cell apoptosis; aging; anagen; apoptotic mitochondrial changes; cell projection organization and biogenesis; cellular response to insulin stimulus; cytoskeleton organization and biogenesis; G1/S-specific positive regulation of cyclin-dependent protein kinase activity; germ cell development; glucose homeostasis; glucose metabolic process; glucose transport; glycogen biosynthetic process; glycogen metabolic process; inflammatory response; insulin receptor signaling pathway; insulin-like growth factor receptor signaling pathway; lipopolysaccharide-mediated signaling pathway; maternal placenta development; myelin maintenance in the peripheral nervous system; negative regulation of apoptosis; negative regulation of autophagy; negative regulation of caspase activity; negative regulation of cell size; negative regulation of fatty acid beta-oxidation; negative regulation of JNK cascade; negative regulation of protein kinase activity; negative regulation of proteolysis; osteoblast differentiation; peptidyl-serine phosphorylation; peptidyl-threonine phosphorylation; phosphorylation; positive regulation of apoptosis; positive regulation of blood vessel endothelial cell migration; positive regulation of cell growth; positive regulation of cellular protein metabolic process; positive regulation of endodeoxyribonuclease activity; positive regulation of endothelial cell proliferation; positive regulation of fat cell differentiation; positive regulation of glucose import; positive regulation of glycogen biosynthetic process; positive regulation of lipid biosynthetic process; positive regulation of nitric oxide biosynthetic process; positive regulation of nitric-oxide synthase activity; positive regulation of peptidyl-serine phosphorylation; positive regulation of proteasomal ubiquitin-dependent protein catabolic process; positive regulation of protein amino acid phosphorylation; positive regulation of transcription factor activity; positive regulation of transcription from RNA polymerase II promoter; positive regulation of vasoconstriction; protein amino acid phosphorylation; protein catabolic process; protein import into nucleus, translocation; protein kinase B signaling cascade; protein ubiquitination; regulation of cell migration; regulation of glycogen biosynthetic process; regulation of myelination; regulation of protein localization; response to DNA damage stimulus; response to food; response to hormone stimulus; response to organic substance; signal transduction; spinal cord development; striated muscle cell differentiation; translation
|NCBI Summary:||This gene encodes the founding member of the Akt serine-threonine protein kinase gene family that also includes Akt2 and Akt3. This kinase is a major downstream effector of the phosphatidylinositol 3-kinase (PI3K) pathway that mediates the effects of various growth factors such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), insulin and insulin-like growth factor I (IGF-I). It is activated through recruitment to cellular membranes by PI3K lipid products and by phosphorylation by 3-phosphoinositide dependent kinase-1. It then further phosphorylates different downstream proteins in response to various extracellular signals and thus plays a pivotal role in mediating a variety of cellular processes, such as glucose metabolism, glycogen biosynthesis, protein synthesis and turn over, inflammatory response, cell survival (anti-apoptosis) and development. Alternatively spliced transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Sep 2009]|
|NCBI GenInfo Identifier:||341940204|
|NCBI Gene ID:||11651|
|NCBI Accession:||P31750. 2|
|UniProt Secondary Accession:||P31750,Q62274, Q6GSA6,|
|UniProt Related Accession:||P31750|
|Molecular Weight:||55,707 Da|
|NCBI Full Name:||RAC-alpha serine/threonine-protein kinase|
|NCBI Synonym Full Names:||thymoma viral proto-oncogene 1|
|NCBI Official Symbol:||Akt1|
|NCBI Official Synonym Symbols:||Akt; PKB; Rac; PKB/Akt; PKBalpha|
|NCBI Protein Information:||RAC-alpha serine/threonine-protein kinase|
|UniProt Protein Name:||RAC-alpha serine/threonine-protein kinase|
|UniProt Synonym Protein Names:||AKT1 kinase; Protein kinase B; PKB; Protein kinase B alpha; PKB alpha; Proto-oncogene c-Akt; RAC-PK-alpha; Thymoma viral proto-oncogene|
|Protein Family:||Potassium channel|
|UniProt Gene Name:||Akt1|
|UniProt Entry Name:||AKT1_MOUSE|
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.
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Mouse PKB were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Mouse PKB were tested on 3 different plates, 20 replicates in each plate.
|Intra-assay Precision||Inter-assay Precision|
|C V (%)||5.91||5.19||4.42||6.07||5.70||3.53|
The recovery of Mouse PKB spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.
|Sample Type||Range (%)||Average Recovery (%)|
|EDTA plasma (n=5)||85-98||91|
|Cell culture media (n=5)||92-108||98|
Samples were spiked with high concentrations of Mouse 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)|
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.
|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.