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Immunoprecipitation of native chromatin: NChIP
Methods 31 (2003) 76–82 www.elsevier.com/locate/ymeth
Immunoprecipitation of native chromatin: NChIP Laura P. OÕNeill* and Bryan M. Turner Chromatin and Gene Expression Group, Anatomy Department, University of Birmingham Medical School, Birmingham B15 2TT, UK Accepted 19 February 2003
Abstract Chromatin immunoprecipitation (ChIP) is widely used in many fields to analyze the distribution of specific proteins, or their modified isoforms, across defined DNA domains. ChIP procedures fall into two main categories, namely, those that use native chromatin prepared by nuclease digestion (designated NChIP), and those that use chromatin in which DNA and proteins are crosslinked, either chemically or with UV light (designated XChIP). Each procedure has its own advantages and drawbacks. Here, we outline the methods currently in use in our laboratory to isolate and immunoprecipitate native chromatin from cultured cells, and to isolate and analyze immunoprecipitated protein and DNA. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Chromatin; Nucleosome; Immunoprecipitation; Modified histones; Anti-histone antibodies; DNA slot blotting
1. Introduction Chromatin immunoprecipitation (ChIP) is a powerful and increasingly widely used technique for analyzing the association of specific proteins, or their modified isoforms, with defined genomic regions. Technically, the approach has two major variants that differ primarily in how the starting (input) chromatin is prepared. The first (here designated NChIP) uses native chromatin prepared by micrococcal nuclease digestion of cell nuclei by standard procedures. Resolution can be increased by using purified mononucleosomes as the input chromatin. This approach was first described by Hebbes et al. [1]. The second variant uses chromatin crosslinked by addition of formaldehyde to growing cells, prior to fragmentation, usually by sonication. This approach (here designated XChIP) was pioneered by Varshavsky and co-workers [2–4] and has been extensively developed and refined since [5–9]. Some workers have used mild formaldehyde crosslinking followed by nuclease digestion [10]. UV irradiation has been successfully employed
*
Corresponding author. Fax: +44-121-414-6815. E-mail address: [email protected] (L.P. OÕNeill).
as an alternative crosslinking technique [11–14]. The intrinsic advantages and disadvantages of the two experimental approaches are summarized in Table 1. For the great majority of nonhistone proteins, XChIP is the only option as they are not retained on the DNA during nuclease digestion of native chromatin. However, proteins such as MeCP2 that bind DNA with high affinity can be readily analyzed by NChIP [15]. For analysis of histones and their modified isoforms, NChIP offers major advantages, particularly in terms of antibody specificity. It is important to remember that most antisera to modified histones are raised against unfixed, synthetic peptide antigens and that the epitopes they recognize may be disrupted or destroyed in formaldehyde crosslinked chromatin, particularly as the crosslinks are likely to involve lysine e-amino groups in the N-terminal tails. This is likely to explain the consistently low efficiency of XChIP protocols. Grunstein and coworkers have addressed this important issue by preparing antisera specifically designed for XChIP [16]. Alternatively the ability of an antiserum to give consistent immunostaining of formaldehyde-fixed cells on tissue sections could be used as a preliminary indicator of suitability for XChIP. In what follows we describe the NChIP methodology currently in use in the Birmingham laboratory.
1046-2023/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S1046-2023(03)00090-2
L.P. OÕNeill, B.M. Turner / Methods 31 (2003) 76–82
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Table 1 Advantages and disadvantages of chromatin immunoprecipitation using native and crosslinked chromatin (NChIP and XChIP, respectively) NChIP
XChIP
Advantages
Disadvantages
Advantages
Disadvantages
Predictable (testable) antibody specificity
Usually not useful for nonhistone proteins
Often inefficient due to epitope disruption
Efficient precipitation (DNA and protein can both be analyzed) Precipitated DNA can be analyzed accurately without PCR amplification
Selective nuclease digestion may bias input chromatin
Applicable to nonhistone proteins binding weakly (or indirectly) to DNA Minimizes histone rearrangement
Nucleosomes may rearrange during digestion
Applicable to organisms where native chromatin is difficult to prepare (e.g., yeast)
May fix transient or fortuitous interactions to give false picture of steady-state levels Starting chromatin often poorly defined with wide range of fragment sizes
2. The method
2.1. Isolation of chromatin from cultured cells
We outline the whole NChIP procedure from the isolation of chromatin (from cultured cells) through the immunoprecipitation step and subsequent DNA and protein analysis. This method is used routinely in our laboratory, primarily for analysis of histones posttranslationally modified at specific residues [17–19]. As analysis of histone modifications becomes increasingly more detailed, it becomes more important than ever to analyze protein after immunoprecipitation to check the specificity of antibodies within the experiment. It is important to show convincingly that the intended target protein has been selectively precipitated, rather than rely only on the stated specificity of the antibody, even when this has been tested by an appropriate technique, such as inhibition ELISA in the case of NChIP. (See White et al. [20] for details.) Nonspecific binding of the antibody to chromatin should also be carefully controlled. Antibodies are always added in excess to ensure complete depletion of the target protein. This may lead to nonspecific binding, which should be carefully monitored using both preimmune sera and a no-antibody control. Affinity purification of antisera [20] also minimizes background and, if possible, should be routinely carried out. The immunoabsorbant now used by many researchers is the commercially available, pure protein A coupled to Sepharose CL-4B. This routinely gives clean results with minimum nonspecific binding, if the washing regime is adjusted for each antiserum used. Washing of the bound material on the immunoabsorbant is critical. We have found that the use of large-volume washes considerably reduces nonspecific binding. The stringency of the washing should be based on the affinity of the antibody for the target protein. In some cases up to 0.5 M NaCl has been used as a final wash but in these circumstances information on the linker histones is lost [21,22]. We have recently begun to explore the use of magnetic beads coated with anti-immunoglobulin antibodies (Dynal, Milltenyi Biotec) rather than protein A– Sepharose to speed up the chromatin binding and washing steps (see below).
To allow us to monitor the yield of chromatin in later steps, and to ensure accuracy in loading slot blots, we routinely label growing cells overnight with [3 H]thymidine to provide an accurate and sensitive marker for bulk DNA. However, if quantitative real-time PCR is set up in the laboratory then [3 H]thymidine may be omitted. This method has been successfully followed on a wide range of cultured cells, including primary embryoid fibroblast, thymocytes, transformed cell lines (HL-60 and HeLa), and undifferentiated and differentiated embryonic stem cells. 1. Harvest cells (5 107 to 1 108 per preparation), wash in PBS containing 5 mM Na butyrate (a deacetylase inhibitor), and resuspend the final cell pellet in 1 TBS (0.15 M NaCl, 0.1 M Tris–HCl, pH 7.5, 3 mM CaCl2 , 2 mM MgCl2 , 5 mM Na butyrate) at a concentration of 2 107 cells/ml. Add an equal volume of 1% Tween 40 diluted in TBS and 1/200th volume of 0.1 M PMSF. Stir the cell suspension on ice for a maximum of 1 h. 2.1.1. Note For some cells a much shorter time in the presence of the detergent is required. For example thymocytes require only 15 min in the presence of the detergent whereas spleen cells require at least an hour. Excessive exposure to the detergent will increase the probability of chromatin reconfiguration and may also lead to bursting of the nuclei and failure of the chromatin extraction. 2. Homogenize in an all-glass homogenizer using a tight pestle and 1 stroke for each 1 ml of suspension. Check the percentage of nuclei present using a hemocytometer. If the preparation retains a significant proportion of intact cells (e.g., >20%), then repeat this step. 3. Centrifuge the cells for 10 min at 600g and resuspend in 25% sucrose/TBS underlayed with a half-volume of 50% sucrose. Centrifuge at 700g for 20 min to pellet the nuclei.
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2.1.2. Note This step can be omitted if the cell numbers are very low (i.e., 5 107 cells or lower). Good clean nuclei are a prerequisite for a successful immunoprecipitation. If the sucrose step is to be omitted, then a high yield of nuclei should be obtained in step 2. Remember to break the surface tension between the 25 and 50% cushion before spinning to prevent the nuclei from becoming trapped at the interface. 4. Resuspend the nuclei in digestion buffer (0.32 sucrose, 50 mM Tris–HCl, pH 7.4, 4 mM MgCl2 , 1 mM CaCl2 , 0.1 M PMSF, 5 mM Na butyrate, 10 mM PMSF). Check the amount of chromatin by measuring the A260 of an aliquot diluted 10-fold in 0.1% SDS (A260 ¼ 1 corresponds to 50 lg/ml of chromatin DNA). Centrifuge at 600g for 10 min and resuspend finally to 0.5 mg/ml. Carry out the micrococcal nuclease digestion in 1-ml aliquots, add 50 U of enzyme/0.5 mg chromatin, and digest for 5 min at 37 °C. 2.1.3. Note This step may have to be refined depending on the cell type being used and the size of the chromatin fragment required at the end. If chromatin is to be used immediately for immunoprecipitations then we normally aim to obtain a chromatin ladder rich in tri-, tetra-, and pentanucleosomes. If, however, we are carrying out the ChIP on mononucleosomes we increase the time of digestion to 7 min to increase the yield of mono- and dinucleosomes in the final preparation. 5. Stop the nuclease reaction by the addition of 0.5 M EDTA to a final concentration of 5 mM and place on ice. Centrifuge the sample for 5 min at 11,600g and retain the first supernatant (S1). Resuspend the pellet in 1 ml of lysis buffer (10 mM Tris, 1 mM EDTA, 5 mM Na butyrate) and dialyze overnight at 4 °C. 6. Centrifuge the samples following dialysis at 2000 rpm for 10 min and determine the amount of material present in S1 and S2 by measuring A260 as above. Resuspend the insoluble material in lysis buffer (1 mM Tris–HCl, pH 7.4, 0.2 mM Na2 EDTA, 0.2 mM PMSF). All samples should be analyzed in the presence of 0.1% SDS by 1.2% agarose gel electrophoresis. Ethidium bromide should not be incorporated into the gel as the SDS present in the sample binds to the ethidium bromide and a poor gel results. Fig. 1 shows the results obtained from a typical chromatin isolation from HL-60 cells.
3. Isolation of mononucleosomes In some cases mononucleosome preparations are used to increase resolution across a chromatin domain.
Fig. 1. Analysis of chromatin fractions by 1.2% agarose gel electrophoresis. Three micrograms of first supernatant (S1), second supernatant (S2), and pellet (P) is loaded onto each track in the presence of SDS (final concentration 0.1%) and electrophoresed for 2 h followed by visualization with ethidium bromide. Size is determined by reference to a 123-bp ladder (Pharmacia M).
We routinely use the following procedure and normally obtain a 10–15% return from the input chromatin. Six 5–25% sucrose linear sucrose gradients (40-ml volume) are prepared in 10 mM Tris–HCl, 0.1 mM Na2 EDTA, 0.1 M NaCl, 25 mM Na butyrate. S1 and S2 are combined and NaCl is added dropwise to a final concentration of 0.1 M. One milliliter of each chromatin preparation is overlain on the top of each gradient and centrifugation performed at 26,000 rpm (x2 t 6:10 1011 ) for approximately 19 h at 4 °C. Fractionation is performed by inserting a long unplugged Pasteur pipet through the gradient and fractions are pumped out from the bottom through a recording spectrophotometer. Fractions (1 ml) are analyzed by 1.25% agarose gel electrophoresis and pooled as appropriate. The degree of purity that should be achieved is shown in Fig. 2. 3.1. Immunoprecipitation of native chromatin Chromatin can be isolated from tissue culture cells (detailed above) or whole tissues by microccoccal nuclease digestion [23]. This procedure generates oligonucleosomes which can range in size from mononucleosomes (if the time of digestion is long) up to 10- to 12-mers if the time of digestion is short. The concentration of micrococcal nuclease can also be used to manipulate the final population of chromatin fragments. The use of freshly prepared chromatin is essential since proteolysis, especially of chromatin isolated from tissues, is well documented. Several workers have noted that following immunoprecipitation from a mixed population of chromatin fragments, the bound fraction preferentially contained higher oligonucleosomes and appeared to be depleted in mononucleosomes [1,17,24]. If this is a
L.P. OÕNeill, B.M. Turner / Methods 31 (2003) 76–82
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Fig. 2. Preparation of mono- and oligonucleosomes by centrifugation of nuclease-digested chromatin through a 5–25% sucrose gradient (left) and analysis of purity by agarose gel electrophoresis (right).
serious problem, then mononucleosomes from sucrose gradients can be employed as starting chromatin, although in our hands the results obtained from either a mixed chromatin population or pure mononucleosomes were the same [17]. The following immunoprecipitation procedure was developed in our laboratory from the protocols of Hebbes et al. [1], and Kamakaka and Thomas [12]. We find it essential to use siliconized Eppendorf tubes and pipet tips to maximize recovery of DNA. This method has some advantages over formaldehyde-fixed chromatin immunoprecipitations or (XChIP) in that both the DNA and protein can routinely be analyzed from a single IP. Furthermore the amount of DNA precipitated is sufficient to allow analysis by accurate DNA slot blotting followed by labeling with radioactive probes [1,17–19] or by real-time PCR [25] or to study allelic differences in patterns of histone modification by PCR-SSCP [15,26]. 1. Add 50–200 ll affinity-purified antibody (50–100 lg immunoglobulin) to 100–200 lg unfixed chromatin and add incubation buffer (50 mM NaCl, 20 mM Tris–HCl, pH 7.5, 20 mM Na butyrate, 5 mM Na2 EDTA, 0.1 mM PMSF) to a final volume of 1 ml. 3.1.1. Note We have found that the use of affinity-purified antibodies reduces the amount of nonspecific binding. The optimum amount of antibody added is dependent on its titer and on the amount of target protein present in the chromatin and must be determined for each antiserum. Commercially available antiserum has been employed successfully in XChIP using very small amounts (5– 50 ll) and subsequent analysis by PCR. We always use affinity purified antisera for ChIP and either whole sera or 50% saturated ammonium sulphate cuts for Western blotting.
2. After overnight incubation (on a very slowly rotating platform) at 4 °C, add 200 ll preswollen protein A– Sepharose (50% v/v slurry, Pharmacia) and continue the incubation for a further 3 h at room temperature. 3.1.2. Notes Protein A–Sepharose is available commercially as a freeze-dried powder and should be preswollen in 50 mM Tris–HCl, 5 mM Na EDTA, 50 mM NaCl. The concentration of the NaCl can be increased depending on the affinity of the antibody for its target protein. Increasing the NaCl concentration reduces the amount of nonspecific binding, but may also reduce the binding affinity of the antibody for the target protein. Preliminary experiments changing the NaCl concentration should be employed to determine the optimum conditions for the antibody used. An alternative to protein A–Sepharose is to use protein A-coated magnetic beads that can be separated on magnetic columns. The immunoprecipitation is set up as in step 1, with the addition of 50 ll protein Acoated magnetic beads directly to the antibody–chromatin mixture. The mixture is incubated on ice for 30 min with occasional gentle mixing. Magnetic separation columns (mColumns, Miltenyi Biotec), on a proprietary stand, are washed with 100 ll of 1% Nonidet P-40 (NP-40) followed by 100 ll Tris–HCl, pH 7.4, before application of the antibody–chromatin sample. Flow through (i.e., material not associated with magnetic beads) is collected (unbound) before washing the column three times with 0.1% NP-40 and elution of the bound material with 2 100 ll 1% SDS heated to 95 °C. Isolation and processing of DNA and protein are then as outlined below. This procedure is faster than the standard protocol and initial results are promising, with acceptable background and reproducibility. Cost,
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however, is considerably increased and column variability is a problem. 3. Centrifuge the antibody–chromatin mixture at 11,600g for 10 min. Carefully remove and keep the supernatant on ice. This is the unbound fraction and should not contain any of the target protein. Resupend the protein A–Sepharose pellet in 1 ml buffer A (50 mM Tris–HCl, pH 7.5, 10 mM EDTA, 5 mM Na butyrate) containing 50 mM NaCl and layer onto 9 ml of the same buffer. After centrifugation at 1200 rpm, for 10 min at 4 °C, remove the supernatant by aspiration and wash the pellet in 10 ml buffer A containing 100 mM NaCl and finally in 10 ml of buffer A containing 150 mM NaCl. 3.1.3. Note The concentration of the NaCl can be increased to reduce nonspecific binding. We have found that large volume washes, carried out in 15-ml siliconized centrifuge tubes, greatly reduce nonspecific binding. 4. Elute the bound material from the protein A–Sepharose by addition of 125 ll 1% SDS in incubation buffer and incubate for 15 min at room temperature with repeated inversion. After centrifugation at 11,600g for 10 min remove the supernatant and store on ice. Repeat this step and combine the two supernatants to give the bound fraction. Add an equal volume of incubation buffer to the bound fraction to reduce the concentration of SDS to 0.5%. Yields of [3 H]T-labeled chromatin from a typical NChIP experiment using antisera to H4 acetylated at defined lysine residues are listed in Table 2. The relative amounts of chromatin immunoprecipitated by the two antisera are consistent with the relative frequencies of H4 isoforms acetylated at lysines 8 and 5 [27]. 3.2. Isolation of DNA Add one-third volume of phenol:chloroform (1:1) to the input, unbound, and bound fractions. Vortex and centrifuge at 600g for 10 min at 4 °C to separate the phases. Remove the supernatant and add an equal vol-
ume of phenol:chloroform. Repeat the centrifugation. Add an equal volume of chloroform, centrifuge as before, and transfer the supernatant to a 6-ml centrifuge tube. Finally precipitate the DNA at 20 °C using 1/ 100th vol of 4 M LiCl and 2 vol of ice-cold ethanol. 3.2.1. Notes We routinely add glycogen (5 lg) as a carrier to maximize the precipitation of DNA from the bound fraction. Ethanol used in the precipitation should be molecular biology grade (e.g., Analar-BDH) and stored at 20 °C to ensure rapid precipitation. Remember to keep the first phenol:chloroform phase for subsequent protein isolation. We routinely analyze the DNA samples by electrophoresis on 1.2% agarose gels followed by staining with ethidium bromide. The amount of DNA in each sample is determined by the amount of [3 H]thymidine present using scintillation counting. 3.3. Isolation of proteins Precipitate the proteins from the first phenol:chlorform phase [28] by addition of 5 lg BSA (carrier), 1/ 100th vol 10 M H2 SO4 , and 12 vol of acetone. After overnight precipitation at 20 °C wash the protein pellets once in acidified acetone (1:6 100 mM H2 SO4 :acetone) and three times in dry acetone. 3.3.1. Notes We routinely analyze the proteins by electrophoresis on SDS–polyacrylamide gels [29]. When proteins are to be analyzed on acid/urea/Triton (AUT) gels [30], the pellet should be resuspended in 500 ll double-distilled H2 O and centrifuged through microconcentrators (Amicon) for 30 min at 11,600g. This step is repeated (to remove residual SDS) and 2 vol of AUT loading buffer (8 M urea, 5% 2-mercaptoethanol, 1 M glacial acetic acid, plus a few drops of tracking dye (pyronineY)) is added to the final concentrated sample. Western blotting and immunostaining are carried out as previously described [27]. Fig. 3 shows results obtained after HL-60
Table 2 Recoveries from a typical NChIP experiment using antibodies to acetylated H4 Antibody to
Chromatin fraction
[3 H]T (103 cpm/10 ll)
Volume (ll)
Total [3 H]T (103 cpm)
% Recovery
H4acK12
Input Unbound Bound
18.90 16.50 3.25
600 250 250
11.3 4.1 0.8
100 36 7
H4acK5
Input Unbound Bound
18.90 21.60 0.52
600 250 250
11.3 5.4 0.13
100 48 1
Pre-immune
Input Unbound Bound
18.90 10.80 0.01
300 250 250
5.6 2.6 0
100 46 0
L.P. OÕNeill, B.M. Turner / Methods 31 (2003) 76–82
Fig. 3. SDS–PAGE and Western blot analysis of immunoprecipitated proteins. Five micrograms of HL-60 histones (C), 20 ll each of the input (IP) and unbound (U) samples, and 80 ll of bound (B) material are added to SDS loading buffer, heated to 100 °C for 10 min, and placed on ice. Following electrophoresis and staining with Coomassie blue (A) the amount of protein in each track is determined using laser densitometry. All subsequent gels for Western blotting are then corrected to give an equal amount of protein in each track. A representative Western blot labeled with an antibody to H4acK8 is shown following immunoprecipitation of chromatin from HL-60 cells using this antibody. A clear depletion of H4acK8 can be seen in the Unbound track and enrichment in the Bound track.
chromatin was immunoprecipitated with H4acK8 and the proteins isolated and analyzed as detailed above. 3.4. DNA analysis 1. [3 H]T-labeled DNA in each sample is determined by scintillation counting. Samples are then diluted in 0.6 M NaCl to give equal counts per milliliter. A 20-ll aliquot is taken from each diluted sample to check the DNA concentration. 2. Following heat denaturation at 95 °C for 10 min and cooling on ice for 5 min, 320 ll of 2 M ammonium acetate is added. A series of five doubling dilutions (250 ll þ 250 ll) in 1 M ammonium acetate are performed and 650 ll of 1 M ammonium acetate is added to every dilution to give a final volume of 900 ll. Two identical slot blots are then prepared using a manifold (Bio-Rad) and Hybond N+ (Amersham), applying 200 ll of each sample in duplicate. Each well is washed twice with 1 M ammonium acetate before the blot is fixed by placing the filter onto a thick layer of filter paper soaked in 0.4 M NaOH for 20 min. 3. Labeling of the filters is performed using standard hybridization techniques. We rountinely prehybridize the filters at 65 °C in 5 SSC, 5 DenhardtÕs, 0.5% SDS for 4 h before adding radioactively labeled DNA probes. Hybridization is performed at 65 °C overnight for random primed probes and 30 °C for end labeled probes. We have also used a kit for rapid hybridization from Amersham. This kit reproducibly gives clean signal-to-background ratios providing unincorporated label is removed from the probe before
81
Fig. 4. Slot-blot analysis of immunoprecipitated DNA. Equal amounts of DNA (based on [3 H]thymidine counts) for input (IP), unbound (U), and bound (B) fractions were blotted onto Hybond Nþ and probed with a probe to the transcriptionally active C-myc gene (1.5-kb SacI fragment) or a probe to an alpha satellite repeat (CAAT), Het266.
hybridization. Filters are then washed with increasing stringency using the following regime: 2 SSC 0.1% SDS, 1 SSC 0.1% SDS, and finally 0.5% SDS. Each wash is performed using prewarmed solution at 65 °C for 20 min. For end-labeled probes the same regime is used but the concentration of SDS is lowered to 0.01% and the temperature to 30 °C. 4. All filters are then wrapped in Saran wrap and exposed to a phosphoimager screen. Signals are analyzed using the Molecular Dynamics Imagequant program and the ratio of signal for the bound (B) and unbound (UB) fractions is determined for each (duplicate) DNA dilution on the blot. These B/UB ratios are then averaged to give the final value (with standard error) for each immunoprecipitation. Fig. 4 shows a typical result obtained following ChIP from HL60 cells with antibody to H4acK8 followed by DNA isolation, slot blotting, and labeling with two different probes. 3.4.1. Note Each slot blot can be labeled at least 10 times before the signal becomes weak and variable.
References [1] T.R. Hebbes, A.W. Thorne, C. Crane-Robinson, EMBO J. 5 (1988) 1395–1402. [2] M.J. Solomon, P.L. Larsen, A. Varshavsky, Cell 53 (1988) 937–947. [3] V. Jackson, Cell 15 (1978) 945–954. [4] M.J. Solomon, A. Varshavsky, Proc. Natl. Acad. Sci. USA 82 (1985) 6470–6474. [5] P.C. Dedon, J.A. Soults, C.D. Allis, M.A. Gorovsky, Anal. Biochem. 197 (1991) 83. [6] V. Orlando, Trends Biochem. Sci. 25 (2000) 99–104. [7] A.L. Clayton, S. Rose, M.J. Barratt, L.C. Mahadevan, EMBO J. 19 (2000) 3714–3726. [8] S. Thomson, A.L. Clayton, L.C. Mahadevan, Mol. Cell. 8 (2001) 1231–1241. [9] V. Orlando, H. Strutt, R. Paro, Methods 11 (1998) 205–214. [10] U.M. Bauer, S. Daujat, S.J. Nielsen, K. Nightingale, T. Kouzarides, EMBO Rep. 1 (2002) 39–44.
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[11] D.S. Gilmour, J.T. Lis, Mol. Cell. Biol. 5 (1985) 2009–2018. [12] R.T. Kamakaka, J.O. Thomas, EMBO J. 9 (1990) 3997– 4006. [13] A.D. Mirzabekov, Gene 135 (1993) 111–118. [14] D. Pruss, S.G. Bavykin, Methods 12 (1997) 36–47. [15] R.I. Gregory, T.E. Randall, C.A. Johnson, S. Khosla, I. Hatada, L.P. OÕNeill, B.M. Turner, R. Feil, Mol. Cell. Biol. 16 (2001) 5426–5436. [16] N. Suka, Y. Suka, A.A. Carmen, J. Wu, M. Grunstein, Mol. Cell (2001) 473–479. [17] L.P. OÕNeill, B.M. Turner, EMBO J. 14 (1995) 3946–3957. [18] C.A. Johnson, L.P. OÕNeill, A. Mitchell, B.M. Turner, Nucleic Acids Res. 26 (1998) 994–1001. [19] L.P. OÕNeill, A.M. Keohane, J.S. Lavender, V. McCabe, E. Heard, P. Avner, N. Brockdorff, B.M. Turner, EMBO J. 18 (1999) 2897–28907. [20] D.A. White, N.D. Belyaev, B.M. Turner, Methods 19 (1999) 417–424.
[21] C.A. Perry, C.A. Dadd, C.D. Allis, A.T. Annuziato, Biochemistry 32 (1993) 13605–13614. [22] A.T. Annuziato, M.B. Eason, C.A. Perry, Biochemistry 34 (1995) 2916–2924. [23] R.D. Kornberg, J.W. LaPointe, Y. Lorch, Methods Enzymol. 170 (1989) 3–14. [24] E. Mendelson, D. Landsman, S. Druckmann, M. Bustin, Eur. J. Biochem. 160 (1986) 253. [25] M.D. Litt, M. Simpwon, M. Gaszner, C.D. Allis, G. Felsenfeld, Science 293 (2001) 2453–2455. [26] R.I. Gregory, R. Feil, Nucleic Acids Res. 27 (1999) e32. [27] B.M. Turner, L.P. OÕNeill, I.M. Allan, FEBS Lett. 253 (1989) 141–145. [28] C.G. Matthews, G.H. Goodwin, E.W. Johns, Nucleic Acids Res. 6 (1979) 167–179. [29] U.K. Laemmli, Nature 227 (1970) 680–685. [30] W.M. Bonner, M.H.P. West, J.D. Stedman, Eur. J. Biochem. 109 (1980) 17–23.
Immunoprecipitation of native chromatin: NChIP Laura P. OÕNeill* and Bryan M. Turner Chromatin and Gene Expression Group, Anatomy Department, University of Birmingham Medical School, Birmingham B15 2TT, UK Accepted 19 February 2003
Abstract Chromatin immunoprecipitation (ChIP) is widely used in many fields to analyze the distribution of specific proteins, or their modified isoforms, across defined DNA domains. ChIP procedures fall into two main categories, namely, those that use native chromatin prepared by nuclease digestion (designated NChIP), and those that use chromatin in which DNA and proteins are crosslinked, either chemically or with UV light (designated XChIP). Each procedure has its own advantages and drawbacks. Here, we outline the methods currently in use in our laboratory to isolate and immunoprecipitate native chromatin from cultured cells, and to isolate and analyze immunoprecipitated protein and DNA. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Chromatin; Nucleosome; Immunoprecipitation; Modified histones; Anti-histone antibodies; DNA slot blotting
1. Introduction Chromatin immunoprecipitation (ChIP) is a powerful and increasingly widely used technique for analyzing the association of specific proteins, or their modified isoforms, with defined genomic regions. Technically, the approach has two major variants that differ primarily in how the starting (input) chromatin is prepared. The first (here designated NChIP) uses native chromatin prepared by micrococcal nuclease digestion of cell nuclei by standard procedures. Resolution can be increased by using purified mononucleosomes as the input chromatin. This approach was first described by Hebbes et al. [1]. The second variant uses chromatin crosslinked by addition of formaldehyde to growing cells, prior to fragmentation, usually by sonication. This approach (here designated XChIP) was pioneered by Varshavsky and co-workers [2–4] and has been extensively developed and refined since [5–9]. Some workers have used mild formaldehyde crosslinking followed by nuclease digestion [10]. UV irradiation has been successfully employed
*
Corresponding author. Fax: +44-121-414-6815. E-mail address: [email protected] (L.P. OÕNeill).
as an alternative crosslinking technique [11–14]. The intrinsic advantages and disadvantages of the two experimental approaches are summarized in Table 1. For the great majority of nonhistone proteins, XChIP is the only option as they are not retained on the DNA during nuclease digestion of native chromatin. However, proteins such as MeCP2 that bind DNA with high affinity can be readily analyzed by NChIP [15]. For analysis of histones and their modified isoforms, NChIP offers major advantages, particularly in terms of antibody specificity. It is important to remember that most antisera to modified histones are raised against unfixed, synthetic peptide antigens and that the epitopes they recognize may be disrupted or destroyed in formaldehyde crosslinked chromatin, particularly as the crosslinks are likely to involve lysine e-amino groups in the N-terminal tails. This is likely to explain the consistently low efficiency of XChIP protocols. Grunstein and coworkers have addressed this important issue by preparing antisera specifically designed for XChIP [16]. Alternatively the ability of an antiserum to give consistent immunostaining of formaldehyde-fixed cells on tissue sections could be used as a preliminary indicator of suitability for XChIP. In what follows we describe the NChIP methodology currently in use in the Birmingham laboratory.
1046-2023/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S1046-2023(03)00090-2
L.P. OÕNeill, B.M. Turner / Methods 31 (2003) 76–82
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Table 1 Advantages and disadvantages of chromatin immunoprecipitation using native and crosslinked chromatin (NChIP and XChIP, respectively) NChIP
XChIP
Advantages
Disadvantages
Advantages
Disadvantages
Predictable (testable) antibody specificity
Usually not useful for nonhistone proteins
Often inefficient due to epitope disruption
Efficient precipitation (DNA and protein can both be analyzed) Precipitated DNA can be analyzed accurately without PCR amplification
Selective nuclease digestion may bias input chromatin
Applicable to nonhistone proteins binding weakly (or indirectly) to DNA Minimizes histone rearrangement
Nucleosomes may rearrange during digestion
Applicable to organisms where native chromatin is difficult to prepare (e.g., yeast)
May fix transient or fortuitous interactions to give false picture of steady-state levels Starting chromatin often poorly defined with wide range of fragment sizes
2. The method
2.1. Isolation of chromatin from cultured cells
We outline the whole NChIP procedure from the isolation of chromatin (from cultured cells) through the immunoprecipitation step and subsequent DNA and protein analysis. This method is used routinely in our laboratory, primarily for analysis of histones posttranslationally modified at specific residues [17–19]. As analysis of histone modifications becomes increasingly more detailed, it becomes more important than ever to analyze protein after immunoprecipitation to check the specificity of antibodies within the experiment. It is important to show convincingly that the intended target protein has been selectively precipitated, rather than rely only on the stated specificity of the antibody, even when this has been tested by an appropriate technique, such as inhibition ELISA in the case of NChIP. (See White et al. [20] for details.) Nonspecific binding of the antibody to chromatin should also be carefully controlled. Antibodies are always added in excess to ensure complete depletion of the target protein. This may lead to nonspecific binding, which should be carefully monitored using both preimmune sera and a no-antibody control. Affinity purification of antisera [20] also minimizes background and, if possible, should be routinely carried out. The immunoabsorbant now used by many researchers is the commercially available, pure protein A coupled to Sepharose CL-4B. This routinely gives clean results with minimum nonspecific binding, if the washing regime is adjusted for each antiserum used. Washing of the bound material on the immunoabsorbant is critical. We have found that the use of large-volume washes considerably reduces nonspecific binding. The stringency of the washing should be based on the affinity of the antibody for the target protein. In some cases up to 0.5 M NaCl has been used as a final wash but in these circumstances information on the linker histones is lost [21,22]. We have recently begun to explore the use of magnetic beads coated with anti-immunoglobulin antibodies (Dynal, Milltenyi Biotec) rather than protein A– Sepharose to speed up the chromatin binding and washing steps (see below).
To allow us to monitor the yield of chromatin in later steps, and to ensure accuracy in loading slot blots, we routinely label growing cells overnight with [3 H]thymidine to provide an accurate and sensitive marker for bulk DNA. However, if quantitative real-time PCR is set up in the laboratory then [3 H]thymidine may be omitted. This method has been successfully followed on a wide range of cultured cells, including primary embryoid fibroblast, thymocytes, transformed cell lines (HL-60 and HeLa), and undifferentiated and differentiated embryonic stem cells. 1. Harvest cells (5 107 to 1 108 per preparation), wash in PBS containing 5 mM Na butyrate (a deacetylase inhibitor), and resuspend the final cell pellet in 1 TBS (0.15 M NaCl, 0.1 M Tris–HCl, pH 7.5, 3 mM CaCl2 , 2 mM MgCl2 , 5 mM Na butyrate) at a concentration of 2 107 cells/ml. Add an equal volume of 1% Tween 40 diluted in TBS and 1/200th volume of 0.1 M PMSF. Stir the cell suspension on ice for a maximum of 1 h. 2.1.1. Note For some cells a much shorter time in the presence of the detergent is required. For example thymocytes require only 15 min in the presence of the detergent whereas spleen cells require at least an hour. Excessive exposure to the detergent will increase the probability of chromatin reconfiguration and may also lead to bursting of the nuclei and failure of the chromatin extraction. 2. Homogenize in an all-glass homogenizer using a tight pestle and 1 stroke for each 1 ml of suspension. Check the percentage of nuclei present using a hemocytometer. If the preparation retains a significant proportion of intact cells (e.g., >20%), then repeat this step. 3. Centrifuge the cells for 10 min at 600g and resuspend in 25% sucrose/TBS underlayed with a half-volume of 50% sucrose. Centrifuge at 700g for 20 min to pellet the nuclei.
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2.1.2. Note This step can be omitted if the cell numbers are very low (i.e., 5 107 cells or lower). Good clean nuclei are a prerequisite for a successful immunoprecipitation. If the sucrose step is to be omitted, then a high yield of nuclei should be obtained in step 2. Remember to break the surface tension between the 25 and 50% cushion before spinning to prevent the nuclei from becoming trapped at the interface. 4. Resuspend the nuclei in digestion buffer (0.32 sucrose, 50 mM Tris–HCl, pH 7.4, 4 mM MgCl2 , 1 mM CaCl2 , 0.1 M PMSF, 5 mM Na butyrate, 10 mM PMSF). Check the amount of chromatin by measuring the A260 of an aliquot diluted 10-fold in 0.1% SDS (A260 ¼ 1 corresponds to 50 lg/ml of chromatin DNA). Centrifuge at 600g for 10 min and resuspend finally to 0.5 mg/ml. Carry out the micrococcal nuclease digestion in 1-ml aliquots, add 50 U of enzyme/0.5 mg chromatin, and digest for 5 min at 37 °C. 2.1.3. Note This step may have to be refined depending on the cell type being used and the size of the chromatin fragment required at the end. If chromatin is to be used immediately for immunoprecipitations then we normally aim to obtain a chromatin ladder rich in tri-, tetra-, and pentanucleosomes. If, however, we are carrying out the ChIP on mononucleosomes we increase the time of digestion to 7 min to increase the yield of mono- and dinucleosomes in the final preparation. 5. Stop the nuclease reaction by the addition of 0.5 M EDTA to a final concentration of 5 mM and place on ice. Centrifuge the sample for 5 min at 11,600g and retain the first supernatant (S1). Resuspend the pellet in 1 ml of lysis buffer (10 mM Tris, 1 mM EDTA, 5 mM Na butyrate) and dialyze overnight at 4 °C. 6. Centrifuge the samples following dialysis at 2000 rpm for 10 min and determine the amount of material present in S1 and S2 by measuring A260 as above. Resuspend the insoluble material in lysis buffer (1 mM Tris–HCl, pH 7.4, 0.2 mM Na2 EDTA, 0.2 mM PMSF). All samples should be analyzed in the presence of 0.1% SDS by 1.2% agarose gel electrophoresis. Ethidium bromide should not be incorporated into the gel as the SDS present in the sample binds to the ethidium bromide and a poor gel results. Fig. 1 shows the results obtained from a typical chromatin isolation from HL-60 cells.
3. Isolation of mononucleosomes In some cases mononucleosome preparations are used to increase resolution across a chromatin domain.
Fig. 1. Analysis of chromatin fractions by 1.2% agarose gel electrophoresis. Three micrograms of first supernatant (S1), second supernatant (S2), and pellet (P) is loaded onto each track in the presence of SDS (final concentration 0.1%) and electrophoresed for 2 h followed by visualization with ethidium bromide. Size is determined by reference to a 123-bp ladder (Pharmacia M).
We routinely use the following procedure and normally obtain a 10–15% return from the input chromatin. Six 5–25% sucrose linear sucrose gradients (40-ml volume) are prepared in 10 mM Tris–HCl, 0.1 mM Na2 EDTA, 0.1 M NaCl, 25 mM Na butyrate. S1 and S2 are combined and NaCl is added dropwise to a final concentration of 0.1 M. One milliliter of each chromatin preparation is overlain on the top of each gradient and centrifugation performed at 26,000 rpm (x2 t 6:10 1011 ) for approximately 19 h at 4 °C. Fractionation is performed by inserting a long unplugged Pasteur pipet through the gradient and fractions are pumped out from the bottom through a recording spectrophotometer. Fractions (1 ml) are analyzed by 1.25% agarose gel electrophoresis and pooled as appropriate. The degree of purity that should be achieved is shown in Fig. 2. 3.1. Immunoprecipitation of native chromatin Chromatin can be isolated from tissue culture cells (detailed above) or whole tissues by microccoccal nuclease digestion [23]. This procedure generates oligonucleosomes which can range in size from mononucleosomes (if the time of digestion is long) up to 10- to 12-mers if the time of digestion is short. The concentration of micrococcal nuclease can also be used to manipulate the final population of chromatin fragments. The use of freshly prepared chromatin is essential since proteolysis, especially of chromatin isolated from tissues, is well documented. Several workers have noted that following immunoprecipitation from a mixed population of chromatin fragments, the bound fraction preferentially contained higher oligonucleosomes and appeared to be depleted in mononucleosomes [1,17,24]. If this is a
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Fig. 2. Preparation of mono- and oligonucleosomes by centrifugation of nuclease-digested chromatin through a 5–25% sucrose gradient (left) and analysis of purity by agarose gel electrophoresis (right).
serious problem, then mononucleosomes from sucrose gradients can be employed as starting chromatin, although in our hands the results obtained from either a mixed chromatin population or pure mononucleosomes were the same [17]. The following immunoprecipitation procedure was developed in our laboratory from the protocols of Hebbes et al. [1], and Kamakaka and Thomas [12]. We find it essential to use siliconized Eppendorf tubes and pipet tips to maximize recovery of DNA. This method has some advantages over formaldehyde-fixed chromatin immunoprecipitations or (XChIP) in that both the DNA and protein can routinely be analyzed from a single IP. Furthermore the amount of DNA precipitated is sufficient to allow analysis by accurate DNA slot blotting followed by labeling with radioactive probes [1,17–19] or by real-time PCR [25] or to study allelic differences in patterns of histone modification by PCR-SSCP [15,26]. 1. Add 50–200 ll affinity-purified antibody (50–100 lg immunoglobulin) to 100–200 lg unfixed chromatin and add incubation buffer (50 mM NaCl, 20 mM Tris–HCl, pH 7.5, 20 mM Na butyrate, 5 mM Na2 EDTA, 0.1 mM PMSF) to a final volume of 1 ml. 3.1.1. Note We have found that the use of affinity-purified antibodies reduces the amount of nonspecific binding. The optimum amount of antibody added is dependent on its titer and on the amount of target protein present in the chromatin and must be determined for each antiserum. Commercially available antiserum has been employed successfully in XChIP using very small amounts (5– 50 ll) and subsequent analysis by PCR. We always use affinity purified antisera for ChIP and either whole sera or 50% saturated ammonium sulphate cuts for Western blotting.
2. After overnight incubation (on a very slowly rotating platform) at 4 °C, add 200 ll preswollen protein A– Sepharose (50% v/v slurry, Pharmacia) and continue the incubation for a further 3 h at room temperature. 3.1.2. Notes Protein A–Sepharose is available commercially as a freeze-dried powder and should be preswollen in 50 mM Tris–HCl, 5 mM Na EDTA, 50 mM NaCl. The concentration of the NaCl can be increased depending on the affinity of the antibody for its target protein. Increasing the NaCl concentration reduces the amount of nonspecific binding, but may also reduce the binding affinity of the antibody for the target protein. Preliminary experiments changing the NaCl concentration should be employed to determine the optimum conditions for the antibody used. An alternative to protein A–Sepharose is to use protein A-coated magnetic beads that can be separated on magnetic columns. The immunoprecipitation is set up as in step 1, with the addition of 50 ll protein Acoated magnetic beads directly to the antibody–chromatin mixture. The mixture is incubated on ice for 30 min with occasional gentle mixing. Magnetic separation columns (mColumns, Miltenyi Biotec), on a proprietary stand, are washed with 100 ll of 1% Nonidet P-40 (NP-40) followed by 100 ll Tris–HCl, pH 7.4, before application of the antibody–chromatin sample. Flow through (i.e., material not associated with magnetic beads) is collected (unbound) before washing the column three times with 0.1% NP-40 and elution of the bound material with 2 100 ll 1% SDS heated to 95 °C. Isolation and processing of DNA and protein are then as outlined below. This procedure is faster than the standard protocol and initial results are promising, with acceptable background and reproducibility. Cost,
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however, is considerably increased and column variability is a problem. 3. Centrifuge the antibody–chromatin mixture at 11,600g for 10 min. Carefully remove and keep the supernatant on ice. This is the unbound fraction and should not contain any of the target protein. Resupend the protein A–Sepharose pellet in 1 ml buffer A (50 mM Tris–HCl, pH 7.5, 10 mM EDTA, 5 mM Na butyrate) containing 50 mM NaCl and layer onto 9 ml of the same buffer. After centrifugation at 1200 rpm, for 10 min at 4 °C, remove the supernatant by aspiration and wash the pellet in 10 ml buffer A containing 100 mM NaCl and finally in 10 ml of buffer A containing 150 mM NaCl. 3.1.3. Note The concentration of the NaCl can be increased to reduce nonspecific binding. We have found that large volume washes, carried out in 15-ml siliconized centrifuge tubes, greatly reduce nonspecific binding. 4. Elute the bound material from the protein A–Sepharose by addition of 125 ll 1% SDS in incubation buffer and incubate for 15 min at room temperature with repeated inversion. After centrifugation at 11,600g for 10 min remove the supernatant and store on ice. Repeat this step and combine the two supernatants to give the bound fraction. Add an equal volume of incubation buffer to the bound fraction to reduce the concentration of SDS to 0.5%. Yields of [3 H]T-labeled chromatin from a typical NChIP experiment using antisera to H4 acetylated at defined lysine residues are listed in Table 2. The relative amounts of chromatin immunoprecipitated by the two antisera are consistent with the relative frequencies of H4 isoforms acetylated at lysines 8 and 5 [27]. 3.2. Isolation of DNA Add one-third volume of phenol:chloroform (1:1) to the input, unbound, and bound fractions. Vortex and centrifuge at 600g for 10 min at 4 °C to separate the phases. Remove the supernatant and add an equal vol-
ume of phenol:chloroform. Repeat the centrifugation. Add an equal volume of chloroform, centrifuge as before, and transfer the supernatant to a 6-ml centrifuge tube. Finally precipitate the DNA at 20 °C using 1/ 100th vol of 4 M LiCl and 2 vol of ice-cold ethanol. 3.2.1. Notes We routinely add glycogen (5 lg) as a carrier to maximize the precipitation of DNA from the bound fraction. Ethanol used in the precipitation should be molecular biology grade (e.g., Analar-BDH) and stored at 20 °C to ensure rapid precipitation. Remember to keep the first phenol:chloroform phase for subsequent protein isolation. We routinely analyze the DNA samples by electrophoresis on 1.2% agarose gels followed by staining with ethidium bromide. The amount of DNA in each sample is determined by the amount of [3 H]thymidine present using scintillation counting. 3.3. Isolation of proteins Precipitate the proteins from the first phenol:chlorform phase [28] by addition of 5 lg BSA (carrier), 1/ 100th vol 10 M H2 SO4 , and 12 vol of acetone. After overnight precipitation at 20 °C wash the protein pellets once in acidified acetone (1:6 100 mM H2 SO4 :acetone) and three times in dry acetone. 3.3.1. Notes We routinely analyze the proteins by electrophoresis on SDS–polyacrylamide gels [29]. When proteins are to be analyzed on acid/urea/Triton (AUT) gels [30], the pellet should be resuspended in 500 ll double-distilled H2 O and centrifuged through microconcentrators (Amicon) for 30 min at 11,600g. This step is repeated (to remove residual SDS) and 2 vol of AUT loading buffer (8 M urea, 5% 2-mercaptoethanol, 1 M glacial acetic acid, plus a few drops of tracking dye (pyronineY)) is added to the final concentrated sample. Western blotting and immunostaining are carried out as previously described [27]. Fig. 3 shows results obtained after HL-60
Table 2 Recoveries from a typical NChIP experiment using antibodies to acetylated H4 Antibody to
Chromatin fraction
[3 H]T (103 cpm/10 ll)
Volume (ll)
Total [3 H]T (103 cpm)
% Recovery
H4acK12
Input Unbound Bound
18.90 16.50 3.25
600 250 250
11.3 4.1 0.8
100 36 7
H4acK5
Input Unbound Bound
18.90 21.60 0.52
600 250 250
11.3 5.4 0.13
100 48 1
Pre-immune
Input Unbound Bound
18.90 10.80 0.01
300 250 250
5.6 2.6 0
100 46 0
L.P. OÕNeill, B.M. Turner / Methods 31 (2003) 76–82
Fig. 3. SDS–PAGE and Western blot analysis of immunoprecipitated proteins. Five micrograms of HL-60 histones (C), 20 ll each of the input (IP) and unbound (U) samples, and 80 ll of bound (B) material are added to SDS loading buffer, heated to 100 °C for 10 min, and placed on ice. Following electrophoresis and staining with Coomassie blue (A) the amount of protein in each track is determined using laser densitometry. All subsequent gels for Western blotting are then corrected to give an equal amount of protein in each track. A representative Western blot labeled with an antibody to H4acK8 is shown following immunoprecipitation of chromatin from HL-60 cells using this antibody. A clear depletion of H4acK8 can be seen in the Unbound track and enrichment in the Bound track.
chromatin was immunoprecipitated with H4acK8 and the proteins isolated and analyzed as detailed above. 3.4. DNA analysis 1. [3 H]T-labeled DNA in each sample is determined by scintillation counting. Samples are then diluted in 0.6 M NaCl to give equal counts per milliliter. A 20-ll aliquot is taken from each diluted sample to check the DNA concentration. 2. Following heat denaturation at 95 °C for 10 min and cooling on ice for 5 min, 320 ll of 2 M ammonium acetate is added. A series of five doubling dilutions (250 ll þ 250 ll) in 1 M ammonium acetate are performed and 650 ll of 1 M ammonium acetate is added to every dilution to give a final volume of 900 ll. Two identical slot blots are then prepared using a manifold (Bio-Rad) and Hybond N+ (Amersham), applying 200 ll of each sample in duplicate. Each well is washed twice with 1 M ammonium acetate before the blot is fixed by placing the filter onto a thick layer of filter paper soaked in 0.4 M NaOH for 20 min. 3. Labeling of the filters is performed using standard hybridization techniques. We rountinely prehybridize the filters at 65 °C in 5 SSC, 5 DenhardtÕs, 0.5% SDS for 4 h before adding radioactively labeled DNA probes. Hybridization is performed at 65 °C overnight for random primed probes and 30 °C for end labeled probes. We have also used a kit for rapid hybridization from Amersham. This kit reproducibly gives clean signal-to-background ratios providing unincorporated label is removed from the probe before
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Fig. 4. Slot-blot analysis of immunoprecipitated DNA. Equal amounts of DNA (based on [3 H]thymidine counts) for input (IP), unbound (U), and bound (B) fractions were blotted onto Hybond Nþ and probed with a probe to the transcriptionally active C-myc gene (1.5-kb SacI fragment) or a probe to an alpha satellite repeat (CAAT), Het266.
hybridization. Filters are then washed with increasing stringency using the following regime: 2 SSC 0.1% SDS, 1 SSC 0.1% SDS, and finally 0.5% SDS. Each wash is performed using prewarmed solution at 65 °C for 20 min. For end-labeled probes the same regime is used but the concentration of SDS is lowered to 0.01% and the temperature to 30 °C. 4. All filters are then wrapped in Saran wrap and exposed to a phosphoimager screen. Signals are analyzed using the Molecular Dynamics Imagequant program and the ratio of signal for the bound (B) and unbound (UB) fractions is determined for each (duplicate) DNA dilution on the blot. These B/UB ratios are then averaged to give the final value (with standard error) for each immunoprecipitation. Fig. 4 shows a typical result obtained following ChIP from HL60 cells with antibody to H4acK8 followed by DNA isolation, slot blotting, and labeling with two different probes. 3.4.1. Note Each slot blot can be labeled at least 10 times before the signal becomes weak and variable.
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