Revvity, 64SKP2TPEG, HTRF Human Total SKP2 Detection Kit, 500 assay points

Overview
S-phase kinase-associated protein 2 (SKP2), also known as FBXL1, is a prominent member of the family of F-box proteins. It functions as an E3 ligase mainly through the formation of an SCF-SKP2 complex. SKP2 is well known for its capacity to regulate the cell cycle. It can ubiquitinate various cell cycle regulators and induce their hydrolysis, including CKIs p21, p27, and p57, as well as the cell cycle protein E (cyclin E) and the cell cycle regulator c-Myc. As a driver of oncogenesis and drug resistance, SKP2 is an attractive target for small-molecule inhibitors and protein degradation strategies aimed at restoring cell cycle checkpoints.
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 readout time or size of assays
How it works
Total SKP2 assay principle
The Total SKP2 assay measures SKP2 levels in cells. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The assay uses 2 antibodies, one labeled with a donor fluorophore and the other with an acceptor. Both antibodies are highly specific for a distinct epitope on the protein.
In the presence of SKP2 this 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 total protein present in the sample and provides a means of assessing the protein's phosphorylation state under a no-wash assay format.

 

Total SKP2 two-plate assay protocol
The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a 384-well low volume detection plate before the addition of Total SKP2 HTRF detection reagents. This protocol allows the cells' viability and confluence to be monitored.

Total SKP2 one-plate assay protocol
Detection of Total SKP2 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 allows miniaturization while maintaining robust HTRF quality. Application-Cell Signaling
Brand-HTRF
Detection Modality-HTRF
Lysis Buffer Compatibility-Lysis Buffer 1 Lysis Buffer 2 Lysis Buffer 4
Molecular Modification-Total
Product Group-Kit
Sample Volume-16 µL
Shipping Conditions-Shipped in Dry Ice
Target-SKP2
Target Class-Phosphoproteins
Target Species-Human
Technology-TR-FRET
Therapeutic Area-Immuno-oncology Oncology
Unit Size-500 assay points