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Mouse RAC-alpha serine/threonine-protein kinase (Akt1) ELISA Kit

SKU:
MOEB0330
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
P31750
Range:
0.312-20 ng/mL
ELISA Type:
Sandwich
Synonyms:
PKB, PKB alpha, PRKBA
Reactivity:
Mouse
€649
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Description

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Product Name:Mouse RAC-alpha serine/threonine-protein kinase (Akt1) ELISA Kit
Product Code:MOEB0330
Alias:RAC-alpha serine/threonine-protein kinase, AKT1 kinase, Protein kinase B, PKB, Protein kinase B alpha, PKB alpha, Proto-oncogene c-Akt, RAC-PK-alpha, Thymoma viral proto-oncogene, Akt1, Akt, Rac, 2.7.11.1
Uniprot:P31750
Reactivity:Mouse
Range:0.312-20 ng/mL
Detection Method:Sandwich
Size:96 Assay
Storage:Please see kit components below for exact storage details
Note:For research use only
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]
UniProt Code:P31750
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
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.

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