The EpiQuik™ MBD2 Binding Activity/Inhibition Assay Ultra Kit is a complete set of optimized reagents designed for measuring the activity/inhibition of MBD2 binding using nuclear extracts or purified MBD2 protein from a broad range of species such as mammalian, plant, fungal, and bacterial, in a variety of forms including, but not limited to, cultured cells and fresh/frozen tissues. The kit has the following advantages and features:
Background InformationMBD2 (methyl-CpG-binding domain protein 2) is a member of the MBD protein family. MBD2 selectively binds to methylated DNA and suppresses transcription from a methylated target gene through recruiting transcriptional repressor complexes. As an epigenetic reader protein to methylated CpG DNA, MBD2 is a novel target that has been accredited for cancer therapy. Some evidence also linked this protein to neurodevelopment. The binding activity of MBD2 to methylated DNA may be affected by MBD2 mutation and biochemical or pharmacological intervention. Selective and potent MBD2 inhibition can induce gene re-expression, as seen with genetic disruption of MBD2 in cancer cells. Thus, detecting binding activity and inhibition of MBD2 is important in elucidating mechanisms of epigenetic regulation of gene activation and silencing, as well as benefiting cancer and neuro disease therapeutics.
Principle & ProcedureIn this assay, methylated DNA is stably coated onto microplate wells. Active MBD2 proteins bind to the methylated DNA. The bound MBD2 proteins can be recognized with specific antibodies. The signal intensity of bound MBD2-antibody complex, which is proportional to binding activity, can then be amplified with enhancer and colorimetrically measured by reading the absorbance in a microplate spectrophotometer at a wavelength of 450 nm. The binding activity of the MBD2 is, in turn, proportional to the optical density intensity measured.
Starting Materials & Input AmountInput materials can be nuclear extracts or purified MBD2 proteins. The amount of nuclear extracts for each assay can be 2 µg to 20 µg with an optimal range of 5-10 µg. The amount of purified proteins can be 10 ng to 500 ng, depending on the purity and catalytic activity of the proteins.