Universal Magnetic Co-IP Kit advantages
- Perform Co-IP of nuclear or cytoplasmic protein complexes, beginning from cells or tissue
- Magnetic beads speed and simplify the procedure while reducing background, enabling the use of low-stringency buffers that maintain complex integrity
- Nuclear extraction procedure removes nuclear protein complexes from the DNA without disrupting protein/protein interactions
- Inhibitor solutions included to preserve the integrity of protein modifications
Perform Co-IP of cytoplasmic & nuclear protein complexes
While Co-IP is often used to study cytoplasmic protein complexes, traditional methods are not optimal for studying DNA-binding proteins because these nuclear complexes are fragile and can be difficult to remove from the DNA. This causes them to either be disrupted during extraction, or to be lost because they remain bound to the DNA. For this reason, in addition to containing components for whole-cell extraction, the Universal Magnetic Co-IP Kit provides nuclear extraction reagents that have been optimized to preserve nuclear protein complexes. The reduced background provided by the low non-specific binding of the kit's magnetic beads makes it possible to use a low-stringency Co-IP/Wash Buffer that helps maintain the protein complexes (Figure 1). To further improve Co-IP of nuclear complexes, the kit’s proprietary Enzymatic Shearing Cocktail uses DNA digestion to release undissociated protein complexes from the DNA. Even histone complexes, which are not released from the DNA by traditional high-salt extraction methods, can be studied (Figure 2).
Figure 1: Co-Immunoprecipitation of a nuclear complex containing SRC-1 and ER?.
The Universal Magnetic Co-IP Kit was used to make nuclear extract from MCF-7 cells that had been induced 1 hour with 10 nM Estradiol. IP was performed on 300 µg samples using 2 µg of SRC-1 pAb, ER? pAb and rabbit IgG (as a negative control). Western blot was then performed using the ER? pAb on 10 µg Input Extract, SRC-1 IP, ER? IP and the rabbit IgG IP.
Figure 2: Detection of acetylated Histone H3.
HeLa nuclear extracts were made using the Universal Magnetic Co-IP Kit and a traditional high-salt extraction protocol, supplemented with 1 µM trichostatin A, a deacetylase inhibitor. Five and ten µg samples of each extract were used in Western with Histone H3 acetyl pAb. Protein was detected only in samples made using the kit’s nuclear extraction procedure, as it was designed to release histone and other protein complexes from DNA while preserving modifications.