Figure 1: Schematic of chromatin immunoprecipitation using ChIP-IT Express.

In ChIP, intact cells are fixed using formaldehyde, which cross-links and therefore preserves protein/DNA interactions. DNA is then sheared into small uniform fragments and the DNA/protein complexes are immunoprecipitated using an antibody directed against the DNA-binding protein of interest. Following immunoprecipitation, the DNA is washed, cross-linking is reversed, and the proteins are removed by Proteinase K treatment. In the original ChIP-IT Kits, the DNA is then rapidly cleaned up using the included DNA purification columns. This DNA purification step in not necessary with ChIP-IT Express (Figure 1). The DNA is then screened to determine which genes were bound by the protein of interest.

Why use ChIP-IT™ Kits?

• Complete solution – all critical reagents needed are supplied
• Direct measurement of protein/DNA interactions, not just histone modifications
• Compatible with genome-wide profiling or selective PCR-based approaches
• No need to optimize reagents and protocol
• No phenol/chloroform extractions – DNA purification columns included in original ChIP-IT Kits, DNA purification step eliminated in ChIP-IT Express

Classically, ChIP has been performed with antibodies directed against abundant chromatin components, such as acetylated histones. While this yields information about transcriptional activity of promoters, it does not reveal which transcription factor is bound to the promoter(s) of interest. In contrast, ChIP using transcription factor-specific antibodies enables direct monitoring of transcription factor/DNA interactions. Unfortunately, this is technically more challenging than classical ChIP, and requires the preparation of several complicated buffers, inhibitor cocktails and blocking reagents. In addition, result validation is difficult without antibodies, controls and a protocol proven to work in ChIP. To overcome these problems and enable ChIP of both transcription factors and histones, Active Motif's ChIP-IT Kits offer a complete solution by providing nearly everything needed to successfully perform ChIP experiments, including positive and negative controls (Figure 2).

Figure 2: Improved chromatin immunoprecipitation using ChIP-IT Express.

A PCR analysis of immunoprecipitated DNA is shown. This figure was selected to demonstrate the efficiency of the ChIP-IT Express Kit. Typically ChIP requires 2 million cells per reaction. However, we have been able to reduce the amount of starting material and observed positive ChIP data from 100,000 cells or less.

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 100,000 and 750,000 cells using a Negative Control IgG and an RNA pol II antibody. The DNA isolated through these ChIP reactions was then analyzed by 36 cycles of PCR using GAPDH positive control primers. (These antibodies and primers are available as the ChIP-IT Control Kit – Human. Kits for mouse and rat are also available.) Ten µl of each PCR was separated on a 1% agarose gel and visualized by UV-illumination following ethidium bromide staining. PCR using the GAPDH primers on DNA isolated with the RNA pol II antibody reproducibly generated more product than similar reactions performed on DNA isolated using the Negative Control IgG.These results demonstrate that ChIP performed with RNA pol II antibody greatly enriched for GAPDH promoter DNA, while ChIP performed with negative IgG did not.