YOUR CART
When performing chromatin immunoprecipitation (ChIP) experiments, it is often useful to prove that two different proteins or histone modifications are present at the same site in the genome. Or, you may want to determine if a protein coincides with a specific histone modification at the same regulatory element. Re-ChIP (aka Sequential ChIP, Chromatin Re-IP and ChIP Re-ChIP) is a relatively new technique that enables sequential chromatin immunoprecipitations to be performed using two different antibodies so that you can assay for the simultaneous presence of two proteins or distinct histone modifications at the same genomic region of interest.
A complete solution for rapid and efficient Re-ChIP
- Convenient magnetic bead-based protocol
- Fewer cells required than other Re-ChIP methods
- Compatible with multi-channel pipetting
Identify protein co-localization in vivo with Re-ChIP-IT
Chromatin immunoprecipitation (ChIP) is extremely useful for studying chromatin biology and transcriptional regulation because it enables the localization of chromatin proteins, modified histones and transcription factors that are bound to specific DNA regions in specific cells. Furthermore, because protein/DNA interactions are fixed while in an endogenous, chromosomal context, ChIP results reflect the influence of chromosomal topology and the effects of cellular regulatory proteins.
The Re-ChIP-IT Kit makes it possible to identify the simultaneous binding of two transcription factors or two histone modifications on a DNA fragment of interest. Chromatin that has been immunoprecipitated is removed from the magnetic beads with a special buffer that prevents the majority of the first antibody from participating in the second IP reaction. The chromatin is then desalted and a second ChIP is performed using a different antibody from the first. The cross-links of these sequentially immunoprecipitated protein-DNA complexes are then reversed and the DNA is analyzed by PCR, similar to conventional ChIP samples.
Figure 1: Sequential chromatin immunoprecipitation using Re-ChIP-IT.
HeLa cells were fixed for 10 minutes with 1% formaldehyde and then chromatin was prepared by sonication shearing (5 pulses). ChIP was performed in duplicate on chromatin isolated from 750,000 cells. DNA was analyzed by PCR using GAPDH positive control primers (36 cycles, Tm = 59˚C). This figure was selected to demonstrate the successful sequential chromatin IP with two different antibodies using the Re-ChIP-IT Kit. The antibodies used are listed above the duplicate lanes.
The lane numbers are the same in each panel to indicate that the DNA is from the same chromatin sample. The left panel shows the results of PCR performed on an aliquot of DNA removed from the experiment after the first ChIP step, the right panel represents PCR results on DNA from chromatin samples after both ChIP steps. For example, chromatin samples subjected to first ChIP using Mouse IgG as a negative control (lanes 1 and 2 in the left panel) were then subjected to a second ChIP with an RNA Pol II antibody (lanes 1 and 2 in the right panel). Chromatin samples in which Mouse IgG was used as either the first antibody (lanes 1 and 2) or second antibody (lanes 5 and 6) show little amplification of GAPDH DNA in either the left (first ChIP) or right panel (first and second ChIP). Chromatin samples in which the first antibody used was anti-RNA Pol II and the second antibody was anti-TFIIB (lanes 3 and 4) show good amplification of GAPDH DNA after the second ChIP (right panel) indicating the co-localization of RNA Pol II and TFIIB at the same region of the GAPDH promoter.
