INSM1 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 from measurement of INSM1 Total in various cell types. Adherent cells were grown to confluence in a T175 flask and were lysed with Lysis Buffer at a density of 500,000 cells/mL. Suspension cells were washed and lysed with 5X Lysis Buffer at a density of 200,000 cells/mL. INSM1 levels were evaluated using the SureFire® Ultra™ assay. Equivalent to approximately 5,000 cells/datapoint for adherent cells or 2,000 cells/datapoint for suspension cells. As expected, Total INSM1 protein was only detected in NCI-H69 lung cancer cells. Negative expression was observed in all cell lines tested in agreement with the literature.