Phospho-Bad (Ser112) Cellular Assay Kit

HTRF® cellular assay kit for measuring endogenous phospho-Bad protein directly in cells

Based on our TR-FRET homogeneous and robust HTRF® technology, the phospho-Bad assay kit is designed for the detection and analysis of dose-response inhibition, as well as for activation studies of endogenous Bad phosphorylated at Ser112, directly in your cell type of interest. Using a streamlined mix-and-read no-wash protocol, this kit is amenable for any use from basic research to all drug screening phases.

This assay also comes as a useful complement of the HTRF cleaved PARP assay, used as a relevant marker of cells undergoing apoptosis.

   Check our lysis buffer compatibility      Check our species compatibility

Assay Principle

The phospho-Bad assay kit can be run with cell lysates or whole cells.

Upon activation, Bcl-2-associated death promoter (Bad) is phosphorylated on Ser112. After cell lysis, phospho-Bad can be detected and studied using the HTRF® kit reagents. This sandwich immunoassay involves two monoclonal antibodies: an anti-phospho-Bad antibody labelled with d2 and an anti-Bad antibody labelled with Eu3+ cryptate. These antibodies may be pre-mixed and added in a single dispensing step for direct detection.

The assay can be run under a two-plate protocol, or be further streamlined to a one-plate assay.

The assays can be run under a two-plate protocol, or be further streamlined to a one-step assay.

Two-plate assay protocol

Cells are plated, stimulated and next lysed in the same 96-well culture plate. Lysates are then transferred to the assay plate for the detection of phosphorylated Bad by HTRF® reagents. This protocol enables the cells' viability and confluence to be monitored.

One-plate assay protocol

Detection of phosphorylated Bad with HTRF® reagents is performed in a single plate used for plating, stimulation, lysis and detection. No washing steps are required. This protocol, designed for HTS, enables miniaturization while maintaining a high quality output.

What to expect at the bench

This video covers the principles of phospho-HTRF assays and gives guidelines for performing them, using our phospho-ERK assay as an example.

Simplified Pathway

Bad is a pro-apoptotic factor which belongs to the Bcl-2 family of proteins and governs mitochondrial membrane permeability by regulating cytochrome C release. In healthy, proliferating cells, Bad is phosphorylated and sequestered in the cytosol by 14-3-3 proteins. In stressed cells, death stimuli induce Bad dephosphorylation and its translocation to the mitochondrial membrane, where it neutralizes Bcl-Xl or BCl-2 by heterodimerization and thus induces cytochrome C release, leading to apoptosis. The activity of Bad is controlled by the MAPK/ERK and the PI3K/AKT signaling pathways. When Bad is phosphorylated by multiple kinases like AKT, p90RSK, p70S6K or PIM, themselves induced by JAK/STAT signaling, it promotes survival of the cell.

Product Performance

1. Western Blot versus HTRF assay

Cos-7 cells were grown in a T175 flask at 37°C, 5% CO2, for 1 day. Day 2: After removal of cell culture medium, 3ml of supplemented lysis buffer were added and incubated for 30 minutes. Soluble supernatants were collected after a 10 minute centrifugation. Equal amounts of lysates were used for a side by side comparison of Western Blot and HTRF®.220 cells can be detected by using HTRF® phospho-Bad (Ser112) wheras1750 cells are needed for the Western Blot. The HTRF® assay is 4-fold more sensitive than Western Blot.

2. PMA dose-response on HEK293 cells

Human HEK293 cells (100,000 cells/well) were stimulated for 30 minutes at 37°C with various concentrations of PMA. After 30 minutes of lysis incubation, phosphorylated Bad was measured using the two-plate assay protocol.

3. Staurosporine inhibition on stimulated MCF7 and Cos-7 cells

Human MCF7 and monkey Cos-7 cells (25,000 cells/well) were incubated for 3 hours at 37°C with various concentrations of Staurosporin inhibitor. Then 0.8 µM of PMA was added for cell stimulation, and sampes were incubated for 30 minutes. After 30 minutes of lysis incubation, inhibition of Bad phosphorylation was measured using the HTRF® Phospho-Bad (Ser112) assay with the two-plate protocol.

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
BAD phospho-S112 kit - 500 tests64BADPEG
BAD phospho-S112 kit - 10,000 tests64BADPEH
BAD phospho-S112 kit -1 x 96 tests64BADPET
BAD phospho-S112 kit - 50,000 tests64BADPEY

Companion products

DescriptionCat. noProduct insertMSDS
BAD phospho-S112 kit control lysate64BADTDA
Phospho-total protein blocking reagent - 2 ml64KB1AAC
Phospho-total protein blocking reagent - 6 ml64KB1AAD
Phospho-total protein lysis buffer #3 - 130 mL64KL3FDF