Assay points.
-
ALSU-PCARP1-A500
500
-
ALSU-PCARP1-A10K
10,000
-
ALSU-PCARP1-A50K
50,000
-
ALSU-PCARP1-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
A549 wild type (WT) and A549 PARP1 knockout (KO, Abcam ab276094) cell lines were cultured to confluence in T75 flasks in 10% FBS containing media. Cells were treated with 1 μM Staurosporine for 4 hours in 1% FBS containing media, lysed in 2 mL of Lysis Buffer, serially diluted with Lysis Buffer, then assayed for Cleaved PARP1 (Asp214) using the SureFire® Ultra™ kit. Assay background is represented by the dotted line. Approximate number of cells/datapoint is indicated.
Data obtained with a 2-plate, 2-incubation protocol. RAW 264.7 cells were seeded at 40K cells/well in a 96 well plate and incubated overnight. Cells were treated with Staurosporine in media containing 1% FBS at the indicated concentrations for 4 hours. Cells were lysed with Lysis Buffer and assayed for Cleaved PARP1 (Asp214) and Total ERK1/2 using respective SureFire® Ultra™ kits. Equivalent to approximately 4,000 cells/datapoint.
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 pre-treated with 1 μM Staurosporine in media containing 1% FBS for 4 hours. Cells were then treated with Olaparib at the indicated concentrations for 10 minutes. Cells were lysed with Lysis Buffer and assayed for Cleaved PARP1 (Asp214) and Total Cofilin using respective SureFire® Ultra™ kits. Equivalent to approximately 4,000 cells/datapoint for Cleaved PARP1 (Asp214) and approximately 200 cells/datapoint for Total Cofilin.
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 Staurosporine in media containing 1% FBS at the indicated concentrations for 4 hours. Cells were lysed with Lysis Buffer and assayed for Cleaved PARP1 (Asp214) and Total Cofilin using respective SureFire® Ultra™ kits. Equivalent to approximately 4,000 cells/datapoint for Cleaved PARP1 (Asp214) and approximately 200 cells/datapoint for Total Cofilin.
Manuals & downloads.
-
PDF
HV Detection Kit Manual
-
PDF
Detection Kit Manual
-
Tech Spec
Technical Data Sheet
Safety Data Sheets
-
Safety Data Sheet (SDS)
Safety Data Sheet EU-EN
-
Safety Data Sheet (SDS)
Safety Data Sheet US