Revvity, 63ADK060PEH, HTRF Human and Mouse Total AMPK Detection Kit, 10,000 Assay Points

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.
HTRF assays offer many advantages over other technologies:

Homogeneous add-and-read format
No wash steps
Low background
Straightforward miniaturization from 96- or 384-well microplates to high density assay formats such as 384-well low volume and 1536-well plates
Stable signal, providing flexibility in time of readout or size of assays
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. 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-10,000 assay points