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. 2006 Jan 5;34(1):e2.
doi: 10.1093/nar/gnj004.

Fast chromatin immunoprecipitation assay

Joel D Nelson  1 , Oleg Denisenko, Pavel Sova, Karol Bomsztyk
Affiliations

Fast chromatin immunoprecipitation assay

Joel D Nelson et al. Nucleic Acids Res. .

Abstract

Chromatin immunoprecipitation (ChIP) is a widely used method to explore in vivo interactions between proteins and DNA. The ChIP assay takes several days to complete, involves several tube transfers and uses either phenol-chlorophorm or spin columns to purify DNA. The traditional ChIP method becomes a challenge when handling multiple samples. We have developed an efficient and rapid Chelex resin-based ChIP procedure that dramatically reduces time of the assay and uses only a single tube to isolate PCR-ready DNA. This method greatly facilitates the probing of chromatin changes over many time points with several antibodies in one experiment.

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Figures

Figure 1
Figure 1
A Chelex-100-based method to isolate PCR-ready DNA from immunoprecipitated chromatin. (A) Anti-K protein antibody (10 µg) was pre-incubated with (+) or without (−) blocking peptide (4 µg) (30 min, RT) (17). Lysates were incubated with the antibodies, complexes were pulled down with protein A beads (IP), and after washing, proteins were eluted by boiling in SDS–PAGE loading buffer. Proteins were resolved by SDS–PAGE and, after transfer to PVDF membranes, immunostained (IS) with anti-K protein antibody. (B) Cells were labeled with 3H-thymidine overnight (10 µCi in 10 ml media). After cross-linking with formaldehyde, cells were lysed and sonicated. Half of the sonicated chromatin was incubated with antibody blocked with the peptide [(+) blocking peptide] and the other half with antibody that was not blocked [(−) blocking peptide]. After five washes with 1 ml of IP buffer, Chelex was added and the mixture was boiled. After cooling, proteinase K (200 µg/ml) was added and the tubes were incubated at 55°C for 60 min, mix was again boiled and beads were centrifuged. 3H counts in the supernatant and the beads were measured using a liquid scintillation counter. (C) Purified rat genomic DNA (Total DNA) was boiled with the Chelex/protein A beads suspension. After cooling to room temperature the suspension was treated with [(+) proteinase K] or without [(−) proteinase K] proteinase K and then the mix was boiled again for 10 min. The suspension was centrifuged and the supernatant was used as a template in real-time PCR using primers to the egr-1 and β-globin genes. Three step real-time PCR was run for 40 cycles. Results are expressed as 40-CT, (Threshold Cycle, Applied Biosystems, ABI7900 manual), which directly reflects levels of amplicons. (D) Sonicated chromatin was incubated with anti-K protein antibody as before. After five washes with 1 ml of IP buffer, Chelex was added and the mixture was boiled. After cooling the mix was treated without or with proteinase K (100 or 200 µg) for 60 min (55°C), suspension was boiled again and the released DNA was used as a template in real-time PCR. Plots show values mean ± SD n = 3.
Figure 2
Figure 2
Verification of the new ChIP protocol. (A) Serum-deprived rat mesangial cells were treated with PMA (10−7 M) for indicated time points. Whole cell RNA was used in RT with random hexamer primers. Real-time PCR was carried out with primers to either egr-1 (exon 1) or LAMC1 (exon 28) genes. Results normalized to β-actin mRNA are shown as fold induction, mean ± SD, two experiments, PCR done in triplicates (top panel). Serum-deprived mesangial cells were treated with PMA as above (top panel). After cross-linking with formaldehyde, cells were lysed, pelleted and sonicated. Chromatin IPs were prepared with either RNA polymerase II (4 µg) (Middle panel) or K protein (10 µg) (Bottom panel) antibody with or without blocking peptides (4 µg). Equal amounts of chromatin fraction were used in the IPs. DNA purified with either the new (solid blue line) or the conventional (dotted blue line) ChIP protocols was used as a template in real-time PCR. The results are expressed as a ratio of the level of PCR products obtained without (−) and with (+) blocking peptide. The graphs show results from two independent IPs done with the new ChIP protocol and results of one representative experiment is shown for the traditional method. PCR was done in triplicates (middle and bottom panel). (B) New ChIP protocol was used to assesses PMA-induced kinetics of RNA polymerase II and K protein recruitment to the different regions (I–VII) along the LAMC1 gene in rat mesangial cells. The graph with results of RT–PCR analysis of mRNA is shown in the right panel (V). Results are are shown as mean values of two independent experiments. Diagram above the graphs represents LAMC1 transcribed (rectangle) and flanking regions (lines). The arrows point at the sites of the respective pair of primers (I and II are 20 and 5 kb 5′ to the start of transcription, respectively, III is the promoter region, IV is exon 2, V is exon 28 and VI and VII are 5 and 20 kb 3′ to the end of the last exon, exon 28). (C) Comparison of the density of RNA polymerase II, K protein and histone H3 in the 5′ flanking (I) and transcribed (II) regions of egr-1, and at the silent β-globin (III) locus. Equal aliquots of sheared chromatin were used in the new ChIP assay with either anti-H3 (4 µg), anti-RNA polymerase II or K antibodies. For H3 ChIP, purified rabbit IgG fraction (4 µg) was used as a mock IP control. Diagram above the graphs represents egr-1 and β-globin genes (rectangle) and flanking regions (lines). The arrows represent the sites of the respective pair of primers (I–III).
Figure 3
Figure 3
Verification of the new ChIP assay in yeast. The new and traditional ChIP methods were used to assess recruitment of the yeast Sir2p to HMR and an adjacent genomic locus. (A) Diagram of the yeast HMR locus (37). Primers for PCR analysis were designed to the indicated regions. (B) Results of ChIP analysis with either antibodies to Sir2p or no antibodies (mock IP). IPs and DNA purification using either the new (blue) or traditional (purple) methods were done in parallel with equal amounts of yeast chromatin. Purified DNA samples were analyzed as above with real-time PCR. Results are expressed as signal ratios of anti-Sir2p IP to mock IP. PCR were done in triplicates, data represented as mean ± SD.
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