Phospho-BAD (Ser112) cellular kit

The Phospho-BAD (Ser112) kit enables the cell-based quantification of phosphorylated BAD (Ser112) as a marker of cells entering apoptosis.
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  • Ease-of-use Ease-of-use
  • High sensitivity High sensitivity
  • Low sample consumption Low sample consumption
The Phospho-BAD (Ser112) kit enables the cell-based quantification of phosphorylated BAD (Ser112) as a marker of cells entering apoptosis.
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Overview

The Phospho-Bad (Ser112) cellular kit is designed for the streamlined and efficient quantification of phosphorylated BAD proteins in cell lysates.This fast and convenient, no-wash protocol offers highly sensitive results and can be applied to all steps of the drug discovery process, from basic research to High Throughput screening.

Benefits

  • COMPATIBLE WITH MANY CELL TYPES
  • SPECIFICITY

Phospho-BAD (Ser112) Assay principle

The Phospho-BAD (Ser112) assay measures BAD when phosphorylated at Ser112. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Phospho-BAD (Ser112) assay uses 2 labeled antibodies: one with a donor fluorophore, the other one with an acceptor. The first antibody is selected for its specific binding to the phosphorylated motif on the protein, the second for its ability to recognize the protein independent of its phosphorylation state. Protein phosphorylation 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 phosphorylated protein present in the sample, and provides a means of assessing the protein’s phosphorylation state under a no-wash assay format.
Phospho-BAD (Ser112) Assay principle

Phospho-BAD (Ser112) 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 adding phospho-BAD (Ser112) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Phospho-BAD (Ser112) 2-plate Assay protocol

Phospho-BAD (Ser112) 1-plate assay protocol

Detection of Phosphorylated BAD (Ser112) 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.
Phospho-BAD (Ser112) 1-plate Assay Protocol

WB versus HTRF assay for Phoshpo-BAD (Ser112) kit

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, 3 mL of supplemented lysis buffer were added and incubated for 30 minutess. 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.
Western Blot versus HTRF assay for Phoshpo-BAD (Ser112) kit

PMA dose-response on HEK293 cells for Phospho-BAD (Ser112) kit

Human HEK293 cells (100,000 cells/well) were stimulated for 30 minutess at 37°C with various concentrations of PMA. After 30 minutess of lysis incubation, phosphorylated Bad was measured using the two-plate assay protocol.
PMA dose-response on HEK293 cells for Phospho-BAD (Ser112) kit

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 minutess. After 30 minutess of lysis incubation, inhibition of Bad phosphorylation was measured using the HTRF Phospho-Bad (Ser112) assay with the two-plate protocol.
Staurosporine inhibition on stimulated MCF7 and Cos-7 cells

Simplified pathway of pro-apoptotic factor Bad signaling

Bad is a pro-apoptotic factor belonging 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 while 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.
Pro-apoptotic factor Bad signaling pathway
Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling

Analyse of PI3K/AKT/mTor translational control pathway - Application Notes

Open R&D: Sanofi Access Platform

In collaboration with Sanofi - Scientific Presentations

Cisbio lysis buffer compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein Assays - Flyers

HTRF cellular phospho-protein assays

Physiologically relevant results fo fast flowing research - Flyers

Save time and money

Switch to HTRF assays - Flyers

Species compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein assays - Flyers

Side-by-side comparison of HTRF, Western Blot, ELISA and AlphaScreen® SureFire®

Do all cell-based kinase assays perform similarly? - Posters

Universal HTRF® phospho-protein platform: from 2D, 3D, primary cells to patient derived tumor cells

Analysis of a large panel of diverse biological samples and cellular models - Posters

HTRF phospho assays reveal subtle drug induced effects in tumor-xenografts

Tumor xenograft analysis: HTRF versus Western blot - Application Notes

HTRF cell-based phospho-protein data normalization

Valuable guidelines for efficiently analyzing and interpreting results - Application Notes

HTRF phospho-total lysis buffer: a universal alternative to RIPA lysis buffers

Increased flexibility of phospho-assays - Application Notes

Best practices for analyzing brain samples with HTRF® phospho assays for neurosciences

Insider Tips for successful sample treatment - Technical Notes

HTRF Alpha-tubulin Housekeeping kit

Properly interpret your compound effect - Application Notes

Optimize your HTRF cell signaling assays on tissues

HTRF and WB compatible guidelines - Technical Notes

Key guidelines to successful cell signaling experiments

Mastering the art of cell signaling assays optimization - Guides

HTRF phospho-assays reveal subtle drug-induced effects

Detailed protocol and direct comparison with WB - Posters

Best practices for analyzing tumor xenografts with HTRF phospho assays

Protocol for tumor xenograft analysis with HTRF - Technical Notes

How to run a cell based phospho HTRF assay

What to expect at the bench - Videos

Unleash the potential of your phosphorylation research with HTRF

Unmatched ease of use, sensitivity and specificity assays - Videos

Product Insert BAD P-S112 Kit / 64BADPEG-64BADPEH

64BADPEG-64BADPEH - Product Insert

Cisbio Product Catalog 2019

All your HTRF assays in one document! - Catalog

A guide to Homogeneous Time Resolved Fluorescence

General principles of HTRF - Guides

How HTRF compares to Western Blot and ELISA

Get the brochure about technology comparison. - Brochures

HTRF® cell signaling platform combined with iCell® Hepatocytes

A solution for phospho-protein analysis in metabolic disorders - Posters

Unleash the potential of your phosphorylation research with HTRF

A fun video introducing you to phosphorylation assays with HTRF - Videos

How to run a cell based phospho HTRF assay

3' video to set up your Phospho assay - Videos

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