Revvity, 64GAPDHPEG, HTRF Human and Mouse GAPDH Housekeeping Kit, 500 Assay Points

Overview
This assay is designed to measure the endogenous expression level of GAPDH in cells and tissues, in order to normalize data obtained on the phospho- and/or total protein(s) of interest. Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) is a key glycolytic enzyme which is constitutively and stably expressed in all cells and tissues. Its amino acid sequence exhibits a high degree of homology between mammalian species. These characteristics make it one of the most commonly used housekeeping proteins. Its concentration is proportional to cell numbers and total protein concentrations, and it is therefore used as an internal control for data normalization, to correct for signal changes caused by experimental variability (e.g. number of cells remaining in the culture plate after treatment, or lysis efficacy). The low sample volume of 4 µL and its compatibility with all HTRF phospho-total protein lysis buffers enable a multi-parametric analysis from the same lysate, with the detection of GAPDH in parallel with the phospho- and/or total protein(s) of interest.
How it works
GAPDH Housekeeping assay principle
The GAPDH Housekeeping assay measures endogenous GAPDH in a cell or tissue lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The GAPDH Housekeeping 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 GAPDH 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 any changes caused by experimental variability under a no-wash assay format.

 

GAPDH Housekeeping 2-plate assay protocol
The assay is run under a two-plate assay protocol, where cells are plated and treated in a culture plate. For the detection step, 4 µL of lysate are subsequently transferred into a low volume 96- or 384-well white plate, and 12 µL of kit diluent are added before the dispensing of 4 µL of HTRF® reagents. This protocol enables monitoring of the cells' viability and confluence in an appropriate cell culture plate. Application-Cell Signaling
Brand-HTRF
Detection Modality-HTRF
Lysis Buffer Compatibility-Lysis Buffer 1 Lysis Buffer 2 Lysis Buffer 3 Lysis Buffer 4
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