Revvity, 64SMAD4TPEG, HTRF Human and Mouse Total SMAD4 Detection Kit, 500 Assay Points

Overview
The Total-SMAD4 cellular assay kit is designed to monitor the expression level of SMAD4, whether phosphorylated or unphosphorylated.
It is compatible with our Phospho-SMAD4 kit, and enables the analysis of phosphorylated and total proteins from a single sample for a better readout of TGF-ß signaling activity.
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-SMAD4 assay principle
The Total-SMAD4 assay quantifies the expression level of SMAD4 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer.
The Total-SMAD4 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 SMAD4 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-SMAD4 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 the addition of the Total-SMAD4 HTRF detection reagents.
This protocol enables the cells' viability and confluence to be monitored.

Total-SMAD4 1-plate assay protocol
Detection of total SMAD4 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
Automation Compatible-Yes
Brand-HTRF
Detection Modality-HTRF
Lysis Buffer Compatibility-Lysis Buffer 1 Lysis Buffer 2 Lysis Buffer 3 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-Cardiovascular Metabolism/Diabetes NASH/Fibrosis Oncology & Inflammation
Unit Size-500 assay points