IRAK1 Total Assay Kit Human

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. 

Data obtained with a 2-plate, 2-incubation protocol. Karpas 299 cells were harvested and seeded in a 96-well plate at 200,000 cells/well and treated with increasing concentrations of JNJ-1013 (IRAK1 PROTAC) for 18 hours. Cells were lysed with Lysis Buffer and assayed separately for IRAK1 and IRAK4 Total using respective SureFire® Ultra™ kits. Equivalent to approximately 20,000 cells/datapoint.

IRAK1 Total levels were assessed in HAP1 wild type (WT) and HAP1 IRAK1 knockout (KO) cells. Cells were cultured to confluency in T175 flasks and lysed in 4 mL of Lysis Buffer. Lysates were serially diluted in Lysis Buffer and IRAK1 Total levels were evaluated using the SureFire® Ultra™ kit. Approximate number of cells/datapoint is indicated.

Sensitivity of the Total IRAK1 assay was assessed by assaying recombinant IRAK1 protein (ab268680). Dilutions of recombinant IRAK1 protein were prepared in Lysis Buffer and evaluated using the SureFire® Ultra™ kit.

Data obtained from measurement of IRAK1 Total in various cell types lysed with Lysis Buffer. Equivalent to approximately 4,000 cells/datapoint for adherent cells or 40,000 cells/datapoint for suspension cells.