HTRF Human & Mouse Total AMPK Detection Kit, 500 Assay Points

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HTRF Human & Mouse Total AMPK Detection Kit, 500 Assay Points

The total AMPK kit is designed to monitor the level of cellular AMP-activated protein kinase and can be used as a normalization assay for the phospho AMPK kit.

For research use only. Not for use in diagnostic procedures. All products to be used in accordance with applicable laws and regulations including without limitation, consumption and disposal requirements under European REACH regulations (EC 1907/2006).

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Feature	Specification
Application	Cell Signaling
Sample Volume	16 µL

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The total AMPK kit is designed to monitor the level of cellular AMP-activated protein kinase and can be used as a normalization assay for the phospho AMPK kit.

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Product Variants

Unit Size: 500 Assay Points

Part #:

63ADK060PEG

List Price

USD 2,147.00

Unit Size: 10,000 Assay Points

Part #:

63ADK060PEH

List Price

USD 12,490.00

Product information

Overview

5' AMP-activated protein kinase (AMPK) is considered the metabolic master switch. AMPK is activated by phosphorylation on Thr172 by CaMKK2 and LKB1, among others. Upon activation, it inhibits synthesis of fatty acids, cholesterol, and triglycerides, and activates fatty acid uptake and ß-oxidation as well as stimulating glucose uptake. AMPK is also involved in autophagy activation through direct phosphorylation of ULK1. Revvity's cell-based homogeneous HTRF kit for total AMPK enables the detection of the AMPK expression level, and is used as a normalization assay with our phospho-AMPK (Thr172) kit.

Specifications

Other Specifications

Application	Cell Signaling
Brand	HTRF
Detection Modality	HTRF
Lysis Buffer Compatibility	Lysis Buffer 1 Lysis Buffer 4 Lysis Buffer 5
Molecular Modification	Total
Product Group	Kit
Sample Volume	16 µL
Shipping Conditions	Shipped in Dry Ice
Target Class	Phosphoproteins
Target Species	Human Mouse
Technology	TR-FRET
Therapeutic Area	Metabolism/Diabetes NASH/Fibrosis
Unit Size	500 Assay Points

Video gallery

How it works

Total-AMPK assay principle

The Total-AMPK assay quantifies the expression level of AMPK in a cell lysate. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Total-AMPK assay uses two labeled antibodies: one coupled to a donor fluorophore, the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of AMPK in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the protein’s expression under a no-wash assay format.

Total-AMPK 2-plate assay protocol

The 2 plate protocol involves culturing cells in a 96-well plate before lysis then transferring lysates to a 384-well low volume detection plate before adding Total AMPK HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Total-AMPK 1-plate assay protocol

Detection of total AMPK with HTRF reagents can be performed in a single plate used for culturing, stimulation and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.

Assay validation

Validation of total AMPK kit in mouse and human cells

Cells were seeded in a T175 flask in complete culture medium and incubated for 2 days until reaching 80% confluency. After treatment with H2O2, cells were lysed and soluble supernatants were collected after a 10 minute centrifugation. Serial dilutions of the cell lysate were performed in the supplemented lysis buffer and 16 µL of each dilution was transferred into a 384-well low volume white microplate before adding 4 µL of the HTRF total-AMPK detection reagents. HTRF signal was recorded after an overnight incubation.

Simplified pathway

AMPK simplified pathway

The Adenosine Mono Phosphate-activated protein Kinase (AMPK) plays a major role in regulating cellular metabolism and metabolic hormones. It is activated when phosphorylated on Thr172. AMPK was originally defined as the upstream kinase for the critical metabolic enzymes Acetyl-CoA carboxylase (ACC1 & ACC2) and HMG-CoA reductase. AMPK stimulates catabolism while inhibiting anabolic pathways and directly affects the hypothalamus to regulate appetite. The regulation of insulin synthesis, particularly its role in glucose transpot, make AMPK an attractive target for metabolic disease research.