MLL3 Complex Chemiluminescent Assay Kit
The MLL3 (KMT2C) Complex Chemiluminescent Assay Kit is designed to measure MLL3 (mixed lineage leukemia protein-3, also known as KMT2C) /WARD complex activity for screening and profiling applications. The MLL3/ WARD complex contains MLL3, WDR5 (WD40 repeat-containing protein 5), Ash2L (Absent, small, homeotic disks-2-like), RbBP5 (retinoblastoma-binding protein 5) and DPY30 (DumPY protein 30). The MLL3 (KMT2C) Complex Chemiluminescent Assay Kit comes in a convenient format, with 8-well strips pre-coated with histone H3 peptide substrate, primary antibody, a secondary HRP-labeled antibody, S-adenosylmethionine, methyltransferase assay buffer, and enough purified MLL3/WARD complex for 100 enzyme reactions.
Figure 1: Illustration of the assay principle.
First, the MLL3 enzyme complex and S-adenosylmethionine are added to pre-coated plate with histone H3 peptide substrate and incubated with or without a test compound. Next, the primary antibody is added. Finally, an HRP-labeled secondary antibody is added, followed by the addition of the HRP substrate to produce chemiluminescence that can be measured using a chemiluminescence reader. Chemiluminescence signal is proportional to the enzymatic activity of the MLL3/WARD complex.
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- TBST Buffer (1 x TBS, pH 8.0, containing 0.05% Tween-20)
- Microplate reader capable of reading chemiluminescence
- Adjustable micropipettor and sterile tips
- Rotating or rocker platform
| Catalog # | Name | Amount | Storage |
| 100323 | MLL3/WDR5/Ash2L/RbBP5/ DPY30 Complex* | 5 µg | -80°C |
| 52120 | 400 µM S-adenosylmethionine | 2 x 250 µl | -80°C |
| 52140Z | Primary Antibody 26 | 10 µl | -80°C |
| 52160 | 4x HMT Assay Buffer 1 | 3 ml | -20°C |
| 52131H | Secondary HRP-Labeled Antibody 2 | 10 µl | -80°C |
| 52100 | Blocking Buffer 4 | 50 ml | +4°C |
| 79670 | ELISA ECL Substrate A (translucent bottle) | 6 ml | Room Temp |
| ELISA ECL Substrate B (brown bottle) | 6 ml | Room Temp | |
| 0.5 M DTT | 200 µl | -20°C | |
| 96-well plate pre-coated with histone substrate | 1 plate | +4°C |
* The concentration of protein is lot-specific and will be indicated on the tube containing the protein.
The Mixed Lineage Leukemia-3 (MLL3, also known as KMT2C) protein belongs to the SET1/MLL family which consists of six (MLL1-4/KMT2A-2D, SET1A/KMT2F, and SET1B/KMT2G) major methyltransferases in mammals. MLL3 is a histone-H3 lysine-4 (H3K4) methyltransferase that promotes H3K4 mono-/di-/tri-methylation, a conserved trait of euchromatin associated with transcriptional activation. MLL3 is involved in regulating cell-type-specific gene expression including those genes encoding DDR and DNA repair proteins. MLL3 forms a complex with RbBP5 (retinoblastoma-binding protein 5), ASH2L (Absent, small, homeotic disks-2-like), WDR5 (WD40 repeat-containing protein 5), and DPY30 (DumPY protein 30) to catalyze methylation of H3K4. WDR5 represents a therapeutically exploitable target for cancer inhibition as it plays a crucial role in MLL3 complex assembly and disassembly. WDR5 has two protein interaction sites: the WDR5-interacting (WIN) binding site and the WDR5-binding-motif (WBM) site. MLL3 forms the complex via WIN binding site, while RbBP5 is bound to WBM site, which is also the site for MYC oncoproteins interaction. The addition of inhibitors that competitively bind to WIN or WBM sites has been shown to disrupt MLL activity as well as to displace MYC from chromatin and therefore disabling its tumorigenic function.
Dillon SC, Zhang X, Trievel RC, Cheng X. Genome Biology 2005; 6:227.
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