AlphaLISA SureFire Ultra

Human and Mouse Total NFκB2 p100/p52 Detection Kit

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Assay points.

  • ALSU-TNFKB2-A500

    500

  • ALSU-TNFKB2-A10K

    10,000

  • ALSU-TNFKB2-A50K

    50,000

  • ALSU-TNFKB2-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. HCT 116 p100 wild type (WT) or knockout (KO, Abcam ab266883) cells were seeded at 80K cells/well in a 96-well plate, incubated overnight and lysed with Lysis Buffer. Lysate was serially diluted and assayed for NFκB2 p100/p52 Total SureFire® Ultra™ assay. Approximate number of cells/datapoint is indicated.

Data obtained with a 2-plate, 2-incubation protocol. HeLa cells were seeded at 40K cells/well in a 96 well plate and incubated overnight. Cells were treated with TNFα at the indicated concentrations for 18 hours. Cells were lysed with Lysis Buffer and lysates were assayed separately for NFκB2 p100 Total and NFκB2 p100/p52 Total using respective SureFire® Ultra™ kits. The ratio of p100 to p100/p52 is indicated at the TNFα concentration of 100 ng/mL. Equivalent to approximately 4,000 cells/datapoint.

Data obtained with a -plate, 2-incubation protocol. RT4 cells were seeded at 40K cells/well in a 96 well plate and incubated overnight. Cells were treated with LIGHT at the indicated concentrations for 24 hours. Cells were lysed with Lysis Buffer and assayed separately for NFκB2 p100 Total and NFκB2 p100/p52 Total using respective SureFire® Ultra™ kits. The ratio of p100 to p100/p52 is indicated at the concentration of LIGHT of 400 ng/mL. Equivalent to approximately 4,000 cells/datapoint.

Manuals & downloads.

Safety Data Sheets

  • Safety Data Sheet (SDS)

    Safety Data Sheet (SDS)

    Safety Data Sheet EU-EN

  • Safety Data Sheet (SDS)

    Safety Data Sheet (SDS)

    Safety Data Sheet US

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