
The AlphaLISA™ SureFire® Ultra™ total Rb assay is a sandwich immunoassay for quantitative detection of Rb (phosphorylated and non-phosphorylated) in cellular lysates using Alpha Technology. This assay is intended to be a normalization phosphorylation studies.
Feature | Specification |
---|---|
Application | Cell Signaling |
Sample Volume | 10 µL |
The AlphaLISA™ SureFire® Ultra™ total Rb assay is a sandwich immunoassay for quantitative detection of Rb (phosphorylated and non-phosphorylated) in cellular lysates using Alpha Technology. This assay is intended to be a normalization phosphorylation studies.
The AlphaLISA SureFire Ultra total Rb assay is a sandwich immunoassay for quantitative detection of total Rb (phosphorylated and non-phosphorylated) in cellular lysates using Alpha Technology.
Formats:
In the AlphaLISA SureFire Ultra assay, Donor beads are coated with streptavidin to capture one of the antibodies, which is biotinylated. Acceptor beads are coated with a proprietary CaptSure™ agent that immobilizes the other antibody, labeled with a CaptSure™ tag. In the presence of target protein, the two antibodies bring the Donor and Acceptor beads close together, generating signal. The amount of light emission is directly proportional to the amount of protein present in the sample.
AlphaLISA SureFire Ultra kits are compatible with:
Alpha SureFire kits can be used for:
Application |
Cell Signaling
|
---|---|
Automation Compatible |
Yes
|
Brand |
AlphaLISA SureFire Ultra
|
Detection Modality |
Alpha
|
Lysis Buffer Compatibility |
Lysis Buffer
|
Molecular Modification |
Total
|
Product Group |
Kit
|
Sample Volume |
10 µL
|
Shipping Conditions |
Shipped in Blue Ice
|
Target |
Rb
|
Target Class |
Phosphoproteins
|
Target Species |
Human
|
Technology |
Alpha
|
Therapeutic Area |
Oncology
|
Unit Size |
50,000 assay points
|
The Total-AlphaLISA SureFire Ultra assay measures the expression level of a protein target in a cell lysate.
The Total-AlphaLISA SureFire Ultra assay uses two antibodies which recognize two different distal epitopes on the targeted protein. AlphaLISA assays require two bead types: Acceptor and Donor beads. Acceptor beads are coated with a proprietary CaptSure™ agent to specifically immobilize the assay specific antibody, labeled with a CaptSure™ tag. Donor beads are coated with streptavidin to capture one of the detection antibodies, which is biotinylated. In the presence of targeted protein, the two antibodies bring the Donor and Acceptor beads in close proximity whereby the singlet oxygen transfers energy to excite the Acceptor bead, allowing the generation of a luminescent Alpha signal. The amount of light emission is directly proportional to the quantity of protein present in the sample.
The two-plate protocol involves culturing and treating the cells in a 96-well plate before lysis, then transferring lysates into a 384-well OptiPlate™ plate before the addition of Total-AlphaLISA SureFire Ultra detection reagents. This protocol permits the cells' viability and confluence to be monitored. In addition, lysates from a single well can be used to measure multiple targets.
Detection of Total target protein with AlphaLISA SureFire Ultra reagents can be performed in a single plate used for culturing, treatment, and lysis. No washing steps are required. This HTS designed protocol allows for miniaturization while maintaining AlphaLISA SureFire Ultra quality.
MCF7 cells were seeded in a 96-well plate (20,000 cells/well) in complete medium, and incubated overnight at 37°C, 5% CO2. The cells were pre-treated with 20 µM Wortmannin in serum free medium for 2 hours, followed by treatment with increasing concentrations of Neuregulin 1 (NRG1) in serum free medium for 24 hours.
After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Rb Total and Phospho (Ser780) levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 2,000 cells) were transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.
NRG1 induced a significant increase in Rb Phospho (Ser780) levels and a modest increase in Rb total.
RT4 cells were seeded in a 96-well plate (40,000 cells/well) in complete medium, and incubated overnight at 37°C, 5% CO2. The cells were treated with increasing concentrations of Palbociclib for 24 hours.
After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Rb Total and Phospho (Ser780) levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 4,000 cells) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.
As expected, Palbociclib triggered a significant decrease in the levels of Rb Phospho (Ser780) and a modest decrease in Rb Total.
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