Revvity, 64MPKPEH, HTRF Human and Mouse Phospho-AMPK (Thr172) Detection Kit, 10,000 Assay Points

Overview
5' AMP-activated protein kinase (AMPK) is considered metabolic master switch. AMPK is activated by phosphorylation on Thr172 by CaMKK2 and LKB1, among ors. Upon activation, it inhibits synsis 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 phospho-AMPK (Thr172) enables quantitative detection of AMP-activated protein Kinase modulation, phosphorylated at Threonine 172.
How it works
Phospho-AMPK (Thr172) assay principle
The Phospho-AMPK (Thr172) assay measures AMPK when phosphorylated at Thr172. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Phospho-AMPK (Thr172) assay uses 2 labeled antibodies: one with a donor fluorophore, the other one with an acceptor. The first antibody is selected for its specific binding to the phosphorylated motif on the protein, the second for its ability to recognize the protein independent of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving both labeled antibodies and which brings the donor fluorophore into close proximity to the acceptor, thereby generating a FRET signal. Its intensity is directly proportional to the concentration of phosphorylated protein present in the sample, and provides a means of assessing the protein's phosphorylation state under a no-wash assay format.

 

Phospho-AMPK (Thr172) 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 Phospho-AMPK (Thr172) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

 

Phospho-AMPK (Thr172) 1-plate assay protocol
Detection of Phosphorylated AMPK (Thr172) 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. Application-Cell Signaling
Brand-HTRF
Detection Modality-HTRF
Lysis Buffer Compatibility-Lysis Buffer 1
Molecular Modification-Phosphorylation
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-10,000 assay points