Mouse Protein Kinase, AMP Activated Alpha 1 (PRKAa1) ELISA Kit

Principle of the Assay

The microtiter plate provided in this kit has been pre-coated with an antibody specific to PRKAa1. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific to PRKAa1. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After the TMB substrate solution is added, only those wells that contain PRKAa1, biotin-conjugated antibody, and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution, and the color change is measured spectrophotometrically at a wavelength of 450nm ± 10nm. The concentration of PRKAa1 in the samples is then determined by comparing the O.D. of the samples to the standard curve.

For Use with serum, plasma, and cell culture supernatants. For Research Use Only. Not for use in diagnostic procedures.

Target Information

Catalytic subunit of AMP-activated protein kinase (AMPK), an energy sensor protein kinase that plays a key role in regulating cellular energy metabolism (By similarity). In response to reduction of intracellular ATP levels, AMPK activates energy-producing pathways and inhibits energy-consuming processes: inhibits protein, carbohydrate and lipid biosynthesis, as well as cell growth and proliferation (By similarity). AMPK acts via direct phosphorylation of metabolic enzymes, and by longer-term effects via phosphorylation of transcription regulators (By similarity). Regulates lipid synthesis by phosphorylating and inactivating lipid metabolic enzymes such as ACACA, ACACB, GYS1, HMGCR and LIPE; regulates fatty acid and cholesterol synthesis by phosphorylating acetyl-CoA carboxylase (ACACA and ACACB) and hormone-sensitive lipase (LIPE) enzymes, respectively (PubMed:15878856). Promotes lipolysis of lipid droplets by mediating phosphorylation of isoform 1 of CHKA (CHKalpha2) (PubMed:34077757). Regulates insulin-signaling and glycolysis by phosphorylating IRS1, PFKFB2 and PFKFB3 (By similarity). AMPK stimulates glucose uptake in muscle by increasing the translocation of the glucose transporter SLC2A4/GLUT4 to the plasma membrane, possibly by mediating phosphorylation of TBC1D4/AS160 (PubMed:16804075, PubMed:16804077). Regulates transcription and chromatin structure by phosphorylating transcription regulators involved in energy metabolism such as CRTC2/TORC2, FOXO3, histone H2B, HDAC5, MEF2C, MLXIPL/ChREBP, EP300, HNF4A, p53/TP53, SREBF1, SREBF2 and PPARGC1A (PubMed:16148943, PubMed:16308421, PubMed:20647423, PubMed:21459323). Acts as a key regulator of glucose homeostasis in liver by phosphorylating CRTC2/TORC2, leading to CRTC2/TORC2 sequestration in the cytoplasm (PubMed:16148943, PubMed:16308421). In response to stress, phosphorylates 'Ser-36' of histone H2B (H2BS36ph), leading to promote transcription (PubMed:20647423). Acts as a key regulator of cell growth and proliferation by phosphorylating TSC2, RPTOR and ATG1/ULK1: in response to nutrient limitation, negatively regulates the mTORC1 complex by phosphorylating RPTOR component of the mTORC1 complex and by phosphorylating and activating TSC2 (PubMed:18439900, PubMed:21258367, PubMed:21205641). In response to nutrient limitation, promotes autophagy by phosphorylating and activating ATG1/ULK1 (PubMed:21258367, PubMed:21205641). In that process also activates WDR45/WIPI4 (By similarity). Phosphorylates CASP6, thereby preventing its autoprocessing and subsequent activation (By similarity). In response to nutrient limitation, phosphorylates transcription factor FOXO3 promoting FOXO3 mitochondrial import (PubMed:23283301). Also acts as a regulator of cellular polarity by remodeling the actin cytoskeleton; probably by indirectly activating myosin (By similarity). AMPK also acts as a regulator of circadian rhythm by mediating phosphorylation of CRY1, leading to destabilize it (PubMed:19833968). May regulate the Wnt signaling pathway by phosphorylating CTNNB1, leading to stabilize it (PubMed:20361929). Also has tau-protein kinase activity: in response to amyloid beta A4 protein (APP) exposure, activated by CAMKK2, leading to phosphorylation of MAPT/TAU; however the relevance of such data remains unclear in vivo (By similarity). Also phosphorylates CFTR, EEF2K, KLC1, NOS3 and SLC12A1 (By similarity).

GENE ID	105787
SWISS PROT	Q5EG47
SYNONYMS	AMPK; AMPKa1; PKA; ACACA kinase; 5'-AMP-Activated Protein Kinase Catalytic Subunit Alpha-1; Acetyl-CoA carboxylase kinase; Hydroxymethylglutaryl-CoA reductase kinase

Materials Supplied

Kit Components	96 Wells Quantity/Size
Pre-coated, ready-to-use 96-well strip plate	1 plate
Plate sealer for 96 wells	2
Standard	2 tubes
Diluent buffer	1 bottle
Detection Reagent A	1 bottle
Detection Reagent B	1 bottle
TMB Substrate	1 tube
Stop Solution	1 tube
Wash Buffer (30 ℅ concentrate)	1 tube
Product data sheet	1 copy

Storage

The TMB Substrate, Wash Buffer (30X concentrate), and the Stop Solution should be stored at 4°C upon receipt, while the other items should be stored at -20°C.

Performance Characteristics

REPEATABILITY	Intra-assay Precision (Precision within an assay): 3 samples with low, middle, and high-level PRKAa1 were tested 20 times on one plate, respectively. Inter-assay Precision (Precision between assays): 3 samples with low, middle, and high-level PRKAa1 were tested on 3 different plates, with 8 replicates in each plate. CV(%) = SD/meanX100 Intra-Assay: CV<10% Inter-Assay: CV<12%
SENSITIVITY	The minimum detectable dose was 30.8pg/mL.
ASSAY RANGE	78.1-5000pg/ml
SPECIFICITY	This assay has high sensitivity and excellent specificity for the detection of PRKAa1. No significant cross-reactivity or interference between PRKAa1 and analogs was observed. Note: Limited by current skills and knowledge, it is impossible to perform all possible cross-reactivity detection tests between PRKAa1 and all analogs, therefore, cross reactivity may still exist.