Clontech’s Protein-DNA Binding Assay provides a safe, fast, and sensitive alternative to traditional electromobility shift assays (EMSA) for detection and quantitative characterization of protein-DNA interactions (1).
- No gel electrophoresis: The binding assay is performed in a 96-well plate, eliminating the need for gel electrophoresis.
- No antibodies, or radioactive labeling: Instead, the assay takes advantage of Clontech’s sensitive and quantitative ProLabel chemiluminescence detection technology.
- Chemiluminescent detection of binding: The ProLabel method consists of fusing a small (~6 kDa) tag to your protein of interest. The resulting ProLabel fusion protein is capable of producing a strong chemiluminescent signal via the ProLabel enzyme complementation assay (2) for direct detection of specific binding between your protein of interest and a dsDNA oligonucleotide.
- Specific and quantitative detection of protein-DNA binding: The assay quantitatively detects specific protein-DNA binding in terms of the ProLabel fusion protein as well as the target sequence.
- Biologically relevant results: Because the ProLabel fusion protein is expressed in mammalian cells, it can acquire biologically relevant posttranslational modifications that may be necessary for functional DNA binding (3).
A Complete Assay System for Cloning, Expression & Detecting Protein-DNA Binding
The assay kit provides the following reagents and materials for cloning and expressing your protein of interest, as well as performing the binding assay, and chemiluminescent detection of protein-DNA binding:
- pProLabel-C Vector, for cloning and expressing your ProLabel fusion protein of interest in mammalian cells
- Specially formulated buffers, for preparing whole cell extracts and performing the binding assay
- Streptavidin-coated 96-well plate, for capturing the protein-DNA complexes
- ProLabel Detection Kit II, which contains a complete set of reagents for measuring ProLabel activity in captured protein-DNA complexes (70 rxns)
- Control vector and oligonucleotides:
- A control vector containing ProLabel fused to a known DNA binding protein
- Biotinylated dsDNA oligonucleotides that serve as positive and negative DNA controls for the binding of the p53 fusion protein control to its cognate DNA consensus element
- Universal In-Fusion Cloning Primers: These primers are designed for directional In-Fusion PCR cloning of inserts from putative yeast one-hybrid clones from any of Clontech’s pGADT7-based cDNA library vectors into the pProLabel-C Vector. These can be used to confirm protein-DNA interactions identified using our Matchmaker Gold Yeast One-Hybrid Library Screening System (Cat. No. 630491). You may also use traditional restriction enzyme-based cloning.
