Assay points.
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ALSU-TP16INK4-A500
500
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ALSU-TP16INK4-A10K
10,000
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ALSU-TP16INK4-A50K
50,000
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ALSU-TP16INK4-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
p16 INK4A (Abcam, Ab84075) and p15 INK4B (Abcam, ab268830) recombinant human proteins were serially diluted with Lysis Buffer and evaluated using the p16 INK4A Total SureFire® Ultra™ kit. No cross-reactivity against p15 INK4B was observed despite sharing over 80% similarity with p16 INK4A.
Data obtained with a 2-plate, 2-incubation protocol. HEK293 cells were seeded at 10K cells/well in a 96 well plate and incubated overnight. Cells were treated with 2.5 μM Palbociclib for 24 hours. Cells were lysed with Lysis Buffer and assayed for Total p16 INK4A using the SureFire® Ultra™ kit. Equivalent to approximately 200 cells/datapoint.
Data obtained from measurement of p16 INK4A Total in various cell types lysed with Lysis Buffer. Approximate number of cells/datapoint is indicated for the various cell lines.
Manuals & downloads.
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PDF
HV Detection Kit Manual
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PDF
Detection Kit Manual
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Tech Spec
Technical Data Sheet
Safety Data Sheets
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Safety Data Sheet (SDS)
Safety Data Sheet EU-EN
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Safety Data Sheet (SDS)
Safety Data Sheet US