Rat AMPK (Phosphorylated Adenosine Monophosphate Activated Protein Kinase) CLIA Kit (RTES00438)

SKU:
RTES00438
Product type:
ELISA
Reactivity:
Rat
ELISA Type:
CLIA
€649

Description

ELISA Kit Technical ManualMSDS

Rat AMPK (Phosphorylated Adenosine Monophosphate Activated Protein Kinase) CLIA Kit

This kit uses Sandwich-CLIA as the method. The micro CLIA plate provided in this kit has been pre-coated with an antibody specific to Rat AMPK . Standards or samples are added to the appropriate micro CLIA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Rat AMPK 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 AMPK, biotinylated detection antibody and Avidin-HRP conjugate will appear fluorescence. The Relative light unit (RLU) value is measured spectrophotometrically by the Chemiluminescence immunoassay analyzer. The RLU value is positively associated with the concentration of Rat AMPK. The concentration of Rat AMPK in the samples can be calculated by comparing the RLU of the samples to the standard curve.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityRat
Detection methodChemiluminescence
Detection range0.63-40 ng/mL
Sensitivity0.38 ng/mL
Sample volume100uL
Sample typeSerum, plasma and other biological fluids
RepeatabilityCV < 15%

Specificity

This kit recognizes Rat AMPK in samples. No significant cross-reactivity or interference between Rat AMPK and analogues was observed.

Typical Data

As the RLU 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)
RLU Average Corrected
4036042
37034
3653836508
2014184
14448
1431614286
106475
6071
62736243
52967
3069
30182988
2.51658
1506
15821552
1.25971
853
912882
0.63584
594
589559
029
31
30--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Rat AMPK were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Rat AMPK were tested on 3 different plates, 20 replicates in each plate.

  Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (ng/mL)1.933.9419.621.803.9120.81
Standard deviation0.220.391.190.220.451.79
C V (%)11.409.906.0712.2211.518.60

Recovery

The recovery of Rat AMPK 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)101-115107
EDTA plasma (n=5)88-10093
Cell culture media (n=5)89-10396

Linearity

Samples were spiked with high concentrations of Rat AMPK 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)
1:2Range (%)85-9996-11183-97
Average (%)9110390
1:4Range (%)85-10199-114103-116
Average (%)92107109
1:8Range (%)102-11599-115103-116
Average (%)108105110
1:16Range (%)96-11189-103102-117
Average (%)10294109

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 CLIA Plate(Dismountable)8 wells X12 strips-20'C, 6 months
Reference Standard2 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 Diluent1 vial, 20 mL4'C, 6 months
Biotinylated Detection Ab Diluent1 vial, 14 mL
HRP Conjugate Diluent1 vial, 14 mL
Concentrated Wash Buffer (25X)1 vial, 30 mL
Substrate Reagent A1 vial, 5 mL4'C (shading light)
Substrate Reagent B1 vial, 5 mL4'C (shading light)
Plate Sealer5 pieces
Product Description1 copy
Certificate of Analysis1 copy

Rat AMPK (Phosphorylated Adenosine Monophosphate Activated Protein Kinase) CLIA Kit (RTES00438) 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 100µL of Substrate mixture solution to each well. Cover with a new plate seal andincubate for no more than 5 min at 37°C. Protect the plate from light.
    11. Determine the RLU value of each well immediately.

Rat AMPK (Phosphorylated Adenosine Monophosphate Activated Protein Kinase) CLIA Kit (RTES00438) Protein Information

UniProt Protein Function:AMPKA2: a catalytic subunit of AMP-activated protein kinase (AMPK). Acts as an energy sensor, playing a key role in regulating cellular energy metabolism. A protein kinase of the CAMKL family whose activation is regulated by the balance between ADP/AMP/ATP, and intracellular Ca(2+) levels. Acts as a metabolic stress-sensing protein kinase switching off biosynthetic pathways when cellular ATP levels are depleted and when 5'-ADP and -AMP rise in response to fuel limitation and/or hypoxia. Activates energy-producing pathways and inhibits energy-consuming processes. Restores ATP levels in cells by switching off anabolic and switching on catabolic pathways. Activated primarily by rising ADP levels and not, as previously thought, solely by AMP. AMPK resembles an adenylate charge regulatory system in which anabolic and catabolic pathways are regulated by adenine nucleotide ratios. Acts via direct phosphorylation of metabolic enzymes and transcription regulators. Regulates fatty acid synthesis by phosphorylating acetyl-CoA carboxylase. Regulates cholesterol synthesis by phosphorylating and inactivating hormone-sensitive lipase and hydroxymethylglutaryl-CoA reductase. Activated by at least two distinct upstream kinases: the tumor suppressor LKB1 and CaMKK2. Also acts as a regulator of cellular polarity by remodeling the actin cytoskeleton, probably by indirectly activating myosin. AMPK is a heterotrimer of an alpha catalytic subunit (AMPKA1 or -2), a beta (AMPKB1 or -2) and a gamma non-catalytic subunit (AMPKG1, -2 or -3). Different possible combinations of subunits give rise to 12 different holoenzymes. Binding of ADP or AMP to non-catalytic gamma subunit (PRKAG1, -2 or -3) results in allosteric activation. AMPK is activated by antihyperglycemic drug metformin, a drug prescribed to patients with type 2 diabetes: in vivo, metformin seems to mainly inhibit liver gluconeogenesis. However, metformin can be used to activate AMPK in muscle and other cells in culture or ex vivo. Selectively inhibited by compound C (6-[4-(2-Piperidin-1-yl-ethoxy)-phenyl)]-3-pyridin-4-yl-pyyrazolo[1,5-a] pyrimidine. Activated by resveratrol, a natural polyphenol present in red wine, and S17834, a synthetic polyphenol. Salicylate/aspirin directly activates kinase activity. Studies in the mouse suggest that AMPK2 may control whole-body insulin sensitivity and is necessary for maintaining myocardial energy homeostasis during ischemia.
UniProt Protein Details:

Protein type:Protein kinase, Ser/Thr (non-receptor); EC 2.7.11.27; EC 2.7.11.1; Kinase, protein; Autophagy; Protein kinase, CAMK; EC 2.7.11.31; CAMK group; CAMKL family; AMPK subfamily

Chromosomal Location of Human Ortholog: 1p31

Cellular Component: nucleoplasm; cytosol

Molecular Function:AMP-activated protein kinase activity; protein serine/threonine kinase activity; protein binding; metal ion binding; [acetyl-CoA carboxylase] kinase activity; protein serine/threonine/tyrosine kinase activity; chromatin binding; histone serine kinase activity; ATP binding; protein kinase activity; [hydroxymethylglutaryl-CoA reductase (NADPH)] kinase activity

Biological Process: lipid biosynthetic process; rhythmic process; cellular lipid metabolic process; carnitine shuttle; glucose homeostasis; signal transduction; protein amino acid phosphorylation; cellular response to glucose starvation; cellular response to nutrient levels; regulation of fatty acid biosynthetic process; regulation of transcription, DNA-dependent; response to stress; cell cycle arrest; positive regulation of autophagy; fatty acid biosynthetic process; mitochondrion organization and biogenesis; negative regulation of TOR signaling pathway; Wnt receptor signaling pathway; transcription, DNA-dependent; organelle organization and biogenesis; regulation of circadian rhythm; cholesterol biosynthetic process; fatty acid homeostasis; positive regulation of glycolysis; insulin receptor signaling pathway; energy reserve metabolic process; autophagy; negative regulation of apoptosis

UniProt Code:Q09137
NCBI GenInfo Identifier:46877068
NCBI Gene ID:5563
NCBI Accession:NP_006243
UniProt Secondary Accession:Q09137,Q9H1E8, Q9UD43,
UniProt Related Accession:P54646,AAB32732
Molecular Weight:62,320 Da
NCBI Full Name:5'-AMP-activated protein kinase catalytic subunit alpha-2
NCBI Synonym Full Names:protein kinase, AMP-activated, alpha 2 catalytic subunit
NCBI Official Symbol:PRKAA2  
NCBI Official Synonym Symbols:AMPK; AMPK2; PRKAA; AMPKa2  
NCBI Protein Information:5'-AMP-activated protein kinase catalytic subunit alpha-2; 5'-AMP-activated protein kinase, catalytic alpha-2 chain; ACACA kinase; AMP-activated protein kinase alpha-2 subunit variant 2; AMP-activated protein kinase alpha-2 subunit variant 3; AMPK alpha 2
UniProt Protein Name:5'-AMP-activated protein kinase catalytic subunit alpha-2
UniProt Synonym Protein Names:Acetyl-CoA carboxylase kinase (EC:2.7.11.27); ACACA kinase; Hydroxymethylglutaryl-CoA reductase kinase (EC:2.7.11.31); HMGCR kinase
UniProt Gene Name:PRKAA2  
UniProt Entry Name:AAPK2_HUMAN
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