Description
The Ikaros zinc-finger (IKZF) family of transcription factors are essential regulators of lymphopoiesis. IKZF1 (Ikaros) and IKZF3 (Aiolos) function as both transcriptional repressors and activators during T and B cell differentiation, as well as in the function of mature cells. Ikaros family proteins partner with multiple complexes, including NuRD, PRC2, and transcription elongation factors. Through these associations, they can modulate gene expression profiles and the chromatin state. the overexpression of these proteins has been linked to sustaining cancer cell proliferation and survival, such as malignant plasma cells and multiple myeloma.
Among the therapeutic strategies investigated to fight cancers, the use of IMIDs (immunomodulatory imide drugs ) and their derivatives, termed CELMoDs (Cereblon E3 Ligase Modulatory Drugs), has become a popular strategy. By binding to CRBN, these compounds induce the ubiquitination of targeted proteins and promote their subsequent degradation. CELMoDs targeting IKZF1 and IKZF3 have shown promising anti-tumor activity in multiple myeloma cell models.
Total IKZF3 assay principle
The HTRF Total IKZF3 assay quantifies the expression level of IKZF3 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based, and does not require gels, electrophoresis, or transfer. The Total IKZF3 assay uses two labeled antibodies, one coupled to a donor fluorophore and the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of IKZF3 in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor, and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the protein’s expression under a no-wash assay format.
Total IKZF3 two-plate assay protocol
The 2-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a 384-well low volume detection plate before the addition of the Total IKZF3 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Total IKZF3 one-plate assay protocol
Detection of Total IKZF3 with HTRF reagents can be performed in a single plate used for culturing, stimulation, and lysis. No washing steps are required. This HTS-designed protocol enables miniaturization while maintaining robust HTRF quality.
IKZF3 Signaling Pathway
Ikaros zinc finger transcription factors are important regulators of the gene programs underlying the development of hematopoietic cell lineages. They recruit different co-factors and interact with target gene loci or chromatin-remodeling complexes. Ikaros mediate different transcriptional outcomes by acting as transcriptional repressors or activators based on cell type.
