The NucBuster Protein Extraction Kit is designed for rapid, efficient preparation of nuclear extracts from mammalian cell cultures (1). The NucBuster Kit is comprised of two proprietary extraction reagents, protease inhibitors, and the reducing agent dithiothreitol (DTT). The entire procedure is performed in one tube without dialysis and the nuclear proteins are ready for assay within 30 minutes. NucBuster extracts are suitable for activity assays and electrophoretic mobility shift assays (EMSA). The NucBuster Kit is designed to maximize the yield of nuclear proteins from diverse cell lines (e.g., CHO-K1, HEK 293, HeLa) and yields highly consistent results without procedural modification.
The NucBuster™ Protein Extraction Kit (Bruggink 2002) provides an alternative to the time intensive and cumbersome traditional methods for preparing nuclear extracts from mammalian cells. Time consuming traditional methods (up to 7 hours), based on a procedure originally described by Dignam et al. (1983), includes suspending cells in hypotonic solution, Dounce homogenization, centrifugation, and dialysis. The NucBuster Kit protocol is rapid and allows for the easy processing of multiple samples. The entire procedure from start to finish yields ready-to-use nuclear extract within 30 minutes. The composition of gentle detergents and salt in the final NucBuster extract is directly compatible with electrophoretic mobility shift assays (EMSA).
The NucBuster protocol is based on two proprietary detergent-based solutions, NucBuster Extraction Reagent 1, optimized for cell lysis and removal of cytoplasmic components, and NucBusterExtraction Reagent 2, optimized for extraction of nuclear proteins. In addition, NucBuster extract is free of the ‘stickiness’ associated with release of genomic DNA, a problem associated with some traditional methods. No Dounce homogenization is required and there is no need for dialysis. The entire procedure is performed in a single microcentrifuge tube and requires only a vortex mixer and microcentrifuge. The kit provides enough reagents for 100 preparations of nuclear extract from 1 x 107 to 5 x 107 cells and the protocol is scalable.