InCELL Hunter Target Engagement Assay | Kinase Binding Assay | 고마바이오텍

특징

• 특정 단백질 표적에 대한 화합물의 결합을 측정하는 cell-based assay kit 입니다.
  
• 세포내에서 진행되므로 화합물의 효능 평가 및 작용 방식을 확인하는데 효과적입니다. 
• EFC 기술을 이용하여, 화합물과 결합한 단백질이 많을 경우 화학발광이 높게 나타납니다.

InCELL Hunter Stabilized Compound-Protein Complex Assay Format

The intracellular target protein in these assays is fused with a small enzyme donor fragment of β-galactosidase (β-gal) called enhanced ProLabel® (ePL). Upon addition of a compound that binds the target, protein levels are stabilized or altered in the cell, and this change can be monitored by measuring target protein abundance using chemiluminescent detection. The detection reagents include a chemiluminescent substrate added with a large enzyme acceptor (EA) fragment that naturally complements with the ePL tag on the target protein to create an active β-gal enzyme. The resulting active enzyme hydrolyzes the substrate to generate a chemiluminescent signal.  The greater the signal corresponds to greater presence of compound-target engagement in the cell.

InCELL Hunter Destabilized Partner Protein Assay Format

In these assays, cell lines are engineered to co-express an untagged target protein and an interacting partner protein fused with ePL. In the absence of an inhibitor compound, the untagged target and ePL-tagged partner protein interact, resulting in stable steady-state levels of ePL-tagged partner protein. Addition of inhibitors that bind the target protein and disrupt the target-partner protein interaction results in ePL-tagged partner protein degradation. The abundance of ePL-tagged partner protein is measured by chemiluminescent detection similar to the format above. The greater the signal corresponds to less compound-target engagement in the cell.

InCELL Pulse Assay Principle

The InCELL Pulse compound-target engagement assays are based on a novel cellular application of the EFC technology incorporating compound binding detection based on protein thermal stability.  Cells expressing a protein of interest fused to the ePL tag are treated with test compound and then subjected to elevated temperatures during a pulse denaturation step. Compound binding protects the target protein from thermal denaturation, which enhances complementation between EA and ePL and increases the chemiluminescent signal measured using the EFC-based detection system. In the absence of compound binding, the target protein forms denatured aggregates that poorly complement with EA, which results in a low chemiluminescent signal.

InCELL Hunter vs. InCELL Pulse

타겟