Human Total KRAS Detection Kit

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. SW1573 cells were seeded at 40K cells/well in a 96 well plate and incubated overnight. Cells were treated with KRAS Degrader-1 or LC-2 PROTAC at the indicated concentrations for 24 hours. Cells were lysed with Lysis Buffer and assayed for Total KRAS, Pan-RAS and ERK using respective SureFire® Ultra™ kits. Equivalent to approximately 4,000 cells/datapoint.

Data obtained with a 2-plate, 2-incubation protocol. SW1573 cells were seeded at 40K cells/well in a 96 well plate and incubated overnight. Cells were treated with AMG-510 inhibitor at the indicated concentrations for 2 hours in media containing 1% FBS. Cells were lysed with Lysis Buffer and assayed for various targets using respective SureFire® Ultra™ kits. Equivalent to approximately 4,000 cells/datapoint.

KRAS wild type, KRAS oncogenic variants G12C, G12D, G12V as well as HRAS and NRAS recombinant proteins were prepared at 1 µg/mL in Lysis Buffer and were evaluated using respective Total KRAS and Pan-RAS SureFire® Ultra™ kits. No cross-reactivity against NRAS and HRAS isoforms was observed.

HCT-116-KRAS wild type (WT) and HCT-116-KRAS knockout (KO, Abcam ab276083) cell lines were cultured to confluence in T175 flasks. Cells were lysed in Lysis Buffer, serially diluted with Lysis Buffer, then assayed separately for Total KRAS and Pan-RAS using respective SureFire® Ultra™ kits. Approximate cells/datapoint is indicated.