Lumit® Anti-Tag Protein Interaction Reagents

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Solution-Based Approach to Detect Protein Interactions of Tagged Proteins

  • Fast and sensitive detection of protein:protein interactions in 30–120 minutes
  • No wash, transfer or immobilization steps
  • Reagents are anti-tag antibodies (and streptavidin) that are conjugated with Lumit® LgBiT and SmBiT
  • Tags include 6His, GST, FLAG®, human IgG and biotin
  • Application Notes available below
  • Early access material; learn more

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Lumit® Anti-Tag Protein Interaction Reagents

Scalable Assay Reagents to Detect PPI of Tagged Proteins

Lumit® Anti-Tag protein-protein interaction (PPI) immunoassay is a homogeneous (no-wash) assay system to measure protein-protein or protein-small molecule interactions involving tagged proteins.

The assay combines immunodetection and NanoLuc® Binary Technology (NanoBiT). NanoBiT is a structural complementation reporter ideal for interaction studies. The NanoBiT® System is composed of two subunits: Large BiT (LgBiT; 18kDa) and Small BiT (SmBiT; 11 amino acid peptide). These subunits can be expressed as recombinant fusions or chemically conjugated to antibodies or other target proteins of interest. The LgBiT and SmBiT subunits have been optimized for stability and minimal self-association due to their weak affinity (Kd = 190µM). When LgBiT and SmBiT are brought into proximity, they form a functional enzyme that generates luminescence in the presence of its substrate.

Lumit® Immunoassay Workflow

Lumit assay principle.

In the Lumit® PPI Immunoassay using protein tags, Streptavidin and antibodies against 6His, GST, FLAG® and human-Fc are labeled with LgBiT or SmBiT. When two proteins with different tags bind, the interaction can be detected using the corresponding SmBiT- and LgBiT-conjugated antibodies.

Lumit Anti-Tag PPI immunoassay principle

Detection of GTP-Dependent KRAS-cRAF Interaction

KRAS interacts with downstream effector cRAF when bound to GTP or non-hydrolysable analogue GppNHp.

Lumit® PPI assays are sensitive enough to detect small changes in GTP-bound state.

KRAS interacts with downstream effector cRAF when bound to GTP or non-hydrolysable analogue GppNHp
Comparison of KRAS-GppNHp and KRAS-GDP binding.
KRAS-GppNHp as percent of total KRAS.

Monitoring VHL-Based Ternary Complex Formation

Compare potency of different PROTACs to target proteins.

Monitoring VHL-based ternary complex formation with a Lumit Anti-Tag PPI assay
Example data comparing potency of PROTACs to target proteins using a Lumit anti-tag assay.
Monitoring  cereblon-based ternary complex formation using a Lumit Anti-Tag PPI assay
Example data comparing potency of PROTACs to target proteins using a Lumit anti-tag assay.

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Petra

Petra

Germany