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AlphaLISA SureFire Ultra

Human Phospho-RIP3 (Ser227) Detection Kit

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Assay points.

  • ALSU-PRIP3-A500

    500

  • ALSU-PRIP3-A10K

    10,000

  • ALSU-PRIP3-A50K

    50,000

  • ALSU-PRIP3-A-HV

    100

Assay Principle

The AlphaLISA™ SureFire® Ultra™ assay enables the rapid and sensitive detection of total and phosphorylated cellular proteins. AlphaLISA™ assays utilize two bead types: Acceptor Beads and Donor Beads. The Acceptor Bead is coated with a proprietary CaptSure™ agent to specifically immobilize the assay specific antibody which is labeled with a CaptSure™ tag. The Donor Bead is coated with streptavidin to capture the biotinylated antibody.  

In the presence of a target protein, the two target-specific antibodies bring Donor and Acceptor Beads into close proximity. When the Donor Beads are activated by a laser (680 nm), singlet oxygen is transferred to the Acceptor Bead leading to the production of an Alpha signal. The amount of light emission (615 nm) from the Acceptor Bead is directly proportional to the amount of target protein present in the sample. If an Acceptor Bead is not in close proximity (i.e. 200 nm) of a Donor bead, little to no signal is produced over background. 

The assay can be executed in a 1-plate or 2-plate assay protocol (Refer to Manual for more details).  

1-plate assay protocol: culturing of cells, treatment, lysis and assay are performed in a single well, enabling miniaturization in high throughput screening programs. 

2-plate assay protocol: cells are cultured and treated in a 96-well culture plate and lysates are then transferred into a separate plate for assay. This format allows the evaluation of multiple targets from a single lysate. 

Technical Specific Data

Data obtained with a 2-plate, 2-incubation protocol. U-937 cells were seeded at 400K cells/well in a 96 well plate then treated with TNFα at the indicated concentrations with 20 µM Z-VAD and 200 nm SM-164 for 7 hours. Cells washed with HBSS, lysed with Lysis Buffer and assayed separately for Phospho (Ser227) and Total RIP3 using respective SureFire® Ultra™ kits. Equivalent to approximately 20,000 cells/datapoint.

Manuals & downloads.

Safety Data Sheets

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