AMPK [pT172] Phospho-ELISA Kit, Human

Catalog number: KHO0651

Novex™  Related applications: Protein Assays and Analysis

Error loading your content!

  Catalog number
Select a plan
Unit size
List price ({{currency}}) Your price ({{currency}}) Availability Qty
{{product.sku}} {{product.sku}}
also known as {{product.formattedSku}} 
{{subscriptionDetails.selectedRatePlan.billingPeriod}} {{subscriptionDetails.selectedRatePlan.billingPeriod}} {{product.availability.message}}
Pro add-ons

Your on-site stock

›› {{supplyCenter.scName}}({{scProduct.stockOnHand}} In stock)
›› {{supplyCenter.scName}}(Out of stock)
›› {{supplyCenter.scName}}
This item is not currently available on-site. Depending on your Supply Center settings you may be able to add the item to cart above else use the Order Non-Stocked Items' tab on the Supply Center home page.
Back to top

Description

The Human AMPK [pT172] ELISA research-use-only kit is to be used for the quantitative determination of AMPK protein phosphorylated at threonine 182 in samples (see sample types indicated) using 96-well plates and a microplate reader. The assay will recognize both natural and recombinant forms of this target.

Performance characteristics

• Sensitivity: <1 Unit/mL
• Standard curve range: 1.6–100 Units/mL
• Sample type(s): cell lysate, tissue homogenate
• Specificity: natural and recombinant human AMPKα [pT172]
• Cross-reactivity: see kit manual for cross-species and/or cross-target reactivity
• Sample volume: 100 μL
• Total assay time: 4 hours

Rigorous validation

Each manufactured lot of this ELISA kit is quality tested for criteria such as sensitivity, specificity, precision, and lot-to-lot consistency. See manual for more information on validation.

Principle of the method

The human AMPK [pT172] solid-phase sandwich ELISA (enzyme-linked immunosorbent assay) is designed to measure the amount of the target bound between a matched antibody pair. A target-specific antibody has been pre-coated in the wells of the supplied microplate. Samples, standards, or controls are then added into these wells and bind to the immobilized (capture) antibody. The sandwich is formed by the addition of the second (detector) antibody, binding to the target on a different epitope from the capture antibody. A conjugated enzyme has been incorporated into the assay. After incubation and washing steps to rid the microplate of unbound substances, a substrate solution is added that reacts with the enzyme-antibody-target complex to produce measurable signal. The intensity of this signal is directly proportional to the concentration of target present in the original specimen.

Target information

AMPK (5’-prime-AMP-activated protein kinase) is a metabolite and stress-sensing kinase that regulates homeostasis, which serves as an energy sensor in all eukaryotic cell types. AMPK, which functions as a serine/threonine protein kinase, exists as a heterotrimeric complex comprised of a catalytic alpha (alpha 1 or alpha 2) subunit and non-catalytic, regulatory beta and gamma (beta 1 or beta 2 and gamma 1, gamma 2, or gamma 3) subunits that differ in their subcellular localization and AMP-dependence. AMPK is phosphorylated and activated by upstream kinases, AMPK kinase (AMPKK) and LKB1. Phosphorylation of Thr-172 in the activation loop of the alpha subunit is a key determinant of AMPK activity. AMPK is activated under conditions that deplete cellular ATP, increasing the AMP/ATP ratio within the cell. These conditions include ischemia, hypoxia, heat shock and glucose deprivation. AMPK is also activated by several hormones and cytokines including leptin and adiponectin and by the anti-diabetic drug metformin.

AMPK causes G1 cell cycle arrest by upregulating the p53-p21 axis. The AMP mimetic, 5-aminoimidaxole-4-carboxamide ribonucleoside (AICAR), which activates AMPK has been shown to cause cell cycle arrest. AMPK activation by AICAR has been reported to inhibit proliferation of cancer cell lines by increasing p21CIP, p27 KIP and p53. Cell cycle arrest by AMPK activation involves accumulation of the tumor suppressor protein p53 by phosphorylation of its serine 15 residue. It is unknown whether p53 is phosphorylated by AMPK directly or by another protein kinase in association with AMPK. The tumor suppressor protein LKB1, a serine/threonine protein kinase upstream of AMPK phosphorylates AMPK at Thr-172 in the activation loop of the kinase domain in the catalytic alpha-subunit. LKB1 associates with p53 and induces upregulation of p21CIP resulting in cell cycle arrest.

AMPK has a role in regulating the mTOR pathway. mTOR is a serine/threonine kinase and is a key regulator of protein synthesis. To inhibit cell growth and protect cells from apoptosis induced by glucose starvation, AMPK phosphorylates TSC2 at Thr-1227 and Ser-1345 increasing the activity of the TSC1-TSC2 complex to inhibit mTOR. In addition, AMPK inhibits mTOR action by phosphorylation on Thr-2446. Thus, AMPK indirectly and directly inhibits the activity of mTOR to limit protein synthesis.

AMPK is also activated by extracellular hormonal stimulation from adiponectin and leptin, peptide homones secreted by adipose tissue. Adiponectin has previously been reported to inhibit vascular smooth muscle cell (SMC) proliferation. Thus, the anti-proliferative effects on vascular cells might be mediated through AMPK. Globular adiponectin activated AMPK in adipocytes and suppressed endothelial cell proliferation. The AMPK activator, AICAR was shown to suppress proliferation of human aortic SMCs. Another AMPK activator, phenformin also inhibited proliferation of both endothelial cells and vascular SMCs.

AMPK plays a role in the cholesterol synthesis pathway. AMPK activation has been reported to phosphorylate and inhibit HMG-CoA reductase, the rate-limiting enzyme in cholesterol synthesis activity thereby decreasing mevalonate synthesis and reducing the products downstream from mevalonate and reducing cholesterol levels. Statins, drugs that are commonly used to decrease cholesterol levels, act by inhibiting HMG-CoA reductase, thereby blocking the conversion of HMG-CoA to mevalonate.

AMPK regulates metabolism by directly phosphorylating rate-limiting enzymes in metabolic pathways involved in fatty acid, cholesterol, and protein synthesis and indirectly controls gene expression.

ELISA kits designed to measure intracellular signaling targets are typically 2–10 times more sensitive than western blotting. The improved sensitivity enables you to detect low-expressing proteins that otherwise may not be distinguishable from background. In addition, the amount of sample needed to run the assay is less than what is needed for western blots.

Related links

Learn more about ELISA kits
Learn more about other immunoassays

For Research Use Only. Not for use in diagnostic procedures.

Specifications

Gene Aliases: AMPK, AMPKa1, MGC33776, MGC57364, AMPK, AMPK2, AMPKa2, PRKAA
Protein Subtype: Serine⁄Threonine Kinases
Accession Number: NP_996790.3, NP_006242.5, NM_006251.5, NM_206907.3, NM_006252.3, NP_006243.2
Shipping Condition: Wet Ice
Marker: AMPK
Gene ID: 5562, 5563
Species: Mouse, Human, Rat
Gene Symbol: PRKAA1, PRKAA2
Sensitivity: <1 U⁄mL
Label or Dye: HRP (Horseradish Peroxidase)
Product Size: 96 assays
Concentration: 1.6 U⁄ml
Sample Volume: 10 µl
Protein Family: Kinases & Inhibitors
Incubation Time: 4 hrs
Detection Method: Colorimetric
Research Category: Signal Transduction
Sample Type (Specific): Cell Extracts
For Use With (Equipment): Microplate Reader

Contents & storage

Pre-coated plate(s), standard or calibrator, detector antibody, HRP conjugate, diluents, wash buffer, substrate(s), and stop solution. Store kit at 2-8°C. See product manual for detailed contents and storage conditions for maximum stability.

Documents

Manuals & protocols

Recommended products