Human Total CD3-Zeta Detection Kit
Opens in a new tabAssay points.
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ALSU-TCD3-A500
500
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ALSU-TCD3-A10K
10,000
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ALSU-TCD3-A50K
50,000
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ALSU-TCD3-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. Jurkat cells were plated at 200K cells/well in a 96 well plate in HBSS + 0.1% BSA, then treated with H2O2 at the indicated concentrations for 10 minutes at 37°C. Cells were lysed with 5X Lysis Buffer and assayed separately for Phospho (Tyr83) and Total CD3 Zeta using respective SureFire® Ultra™ kits. Equivalent to approximately 8,000 cells/datapoint.
Data obtained with a 2-plate, 2-incubation protocol. Jurkat cells were plated at 5×106 cells/mL in 12 well plates in HBSS + 0.1% BSA. Cells were treated for 5 minutes with 10 μg/mL of anti-CD3 Ab (BD cat# 555329) or a mouse IgG isotype control. Cells were lysed with the addition of 5X Lysis Buffer and lysates were further diluted in Lysis Buffer before assayed for Phospho (Tyr83) and Total CD3 Zeta. Equivalent to approximately 8,000 cells/datapoint.
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