Revvity, 64HDAC5TPEG, HTRF Human and Mouse Total HDAC5 Detection Kit, 500 Assay Points

Overview
Histone deacetylases (HDACs) regulate chromatin remodeling and subsequent gene transcription by controlling the status of histone acetylation. Histone deacetylation induces a condensed chromatin conformation, contributing to the repression of gene transcription, which is involved in diverse physiological processes.
HDAC5 belongs to class IIa HDACs, including HDAC4, HDAC7 and HDAC9. It plays a key role in the control of cellular proliferation, differentiation, migration, apoptosis and metabolism.
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 HDAC5 assay principle
The HTRF Total HDAC5 assay quantifies the expression level of HDAC5 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total HDAC5 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 HDAC5 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 HDAC5 2-plate assay protocol
The 2-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring the lysates into a 384-well low volume detection plate before the addition of Total HDAC5 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

 

Total HDAC5 1-plate assay protocol
Detection of Total HDAC5 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 2 Lysis Buffer 3 Lysis Buffer 4
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
Unit Size-500 assay points