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Chromatin Affinity Purification (ChAP) from Arabidopsis thaliana Rosette Leaves Using in vivo Biotinylation System

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The Plant Cell
Apr 2017



Chromatin Affinity Purification (ChAP) is widely used to study chromatin architecture and protein complexes interacting with DNA. Here we present an efficient method for ChAP from Arabidopsis thaliana rosette leaves, in which in vivo biotinylation system is used. The chromatin is digested by Micrococcal Nuclease (MNase), hence the distribution of nucleosomes is also achieved. The in vivo biotinylation system was initially developed for Drosophila melanogaster (Mito et al., 2005), but the presented protocol has been developed specifically for Arabidopsis thaliana (Sura et al., 2017).

Keywords: Chromatin affinity purification (染色质亲和纯化), ChAP-qPCR (ChAP-qPCR), Chromatin immunoprecipitation (染色质免疫沉淀), ChIP (ChIP), Chromatin MNase digestion (染色质MNase消化), Nucleosome occupancy (核小体占位), Histone distribution (组蛋白分布), Arabidopsis thaliana (拟南芥)


Chromatin Immunoprecipitation (ChIP) became one of the most important and commonly used technique to study chromatin structure and organization. However, it requires good-quality antibodies, which will not cross-react with non-specific targets. This is relatively difficult to achieve in plants, which contain cell wall and are rich in photosynthesis-related compounds and proteins frequently causing cross-reactivity problems. On the other hand, obtaining stable transgenic organisms is a routine and easy strategy in plants. For these reasons most plant researchers choose gene tagging, where fusion proteins are obtained and used to study chromatin in an approach alternative to ChIP, that is Chromatin Affinity Purification (ChAP). The ChAP technique has proven to be extremely effective in plant chromatin studies (Zentner and Henikoff, 2014). Moreover, it is usually cheaper than classical ChIP as it does not require generation of antibodies, and is often more effective than ChIP as tags are recognized with higher affinity than antibodies raised directly against proteins of interests. One disadvantage of ChAP is that it cannot be used to study post-translational histone modifications. In the presented protocol, proteins are tagged with a short, Biotin ligase recognition peptide (BLRP), which is in vivo biotinylated by Escherichia coli BirA biotin ligase (de Boer et al., 2003). Consequently, the tagged protein is purified using streptavidin-based purification systems (e.g., Dynabeads M-280 Streptavidin, Invitrogen). As streptavidin has an extraordinarily high affinity for biotin (dissociation constant on the order of 10-14 mol/L), the binding of biotin to streptavidin is one of the strongest non-covalent interactions known in nature (Green, 1975). Alternatively, the presented protocol can be successfully applied for ChAP of proteins labeled with other tags (e.g., MYC, GFP, HA, FLAG) if a suitable system for final purification is used.

Materials and Reagents

  1. Standard pipette tips
  2. 15 ml and 50 ml conical centrifuge tubes
  3. Nylon mesh (pore size 80 μm, Membrane Solutions, catalog number: MENY090080 )
  4. Paper towels
  5. Miracloth (Merck, Calbiochem, catalog number: 475855 )
  6. 1.5-2.0 ml microcentrifuge tubes
  7. 2 ml and 1.5 ml low retention tubes (Maxymum recovery microtubes; Corning, Axygen®, catalog numbers: MCT-200-L-C and MCT-150-L-C )
  8. Plant material: A. thaliana plants expressing E. coli BirA biotin ligase under control of a strong promoter (e.g., Arabidopsis thaliana Act8) with a gene of interest fused with Biotin ligase recognition peptide (BLRP) (de Boer et al., 2003; Mito et al., 2005)
    Note: Plants could be at different developmental stages from one-week-old seedlings to adult, prior flowering. In general, the younger material, the higher efficiency of chromatin isolation. Moreover, younger plants provide higher homogeneity of the tissue used for chromatin extraction and lower variation between replicates. BirA biotin ligase could be expressed from different promoters, however, expression should be kept at high levels. Other possible promoters include Ubiquitin 10 promoter (UBQ10) and Cauliflower Mozaic Virus (CaMV) 35S promoter. Inducible systems e.g., dexamethasone (DEX)-inducible, can be potentially also used, however this was not tested in our laboratory.
  9. 2 M glycine (made of Glycine, Sigma-Aldrich, catalog number: 33226 )
    Note: This product has been discontinued.
  10. Liquid nitrogen, ice
  11. 1 M CaCl2 (made of CaCl2, Sigma-Aldrich, catalog number: C5670 )
  12. Micrococcal nuclease (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: EN0181 )
  13. 0.5 M EGTA (made of EGTA, BioShop, catalog number: EGT101 )
  14. 0.5 M EDTA (made of EDTA, BioShop, catalog number: EDT001 )
  15. RNase A (Sigma-Aldrich, catalog number: R4642 )
  16. Proteinase K (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: EO0491 )
  17. 3 M sodium acetate pH 5.2 (made of CH3COONa, INC Biomedicals Inc.)
  18. Glycogen (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: R0561 )
  19. Ethanol (Avantor Performance Materials, catalog number: BA6420113 )
  20. Agarose (BioShop, catalog number: AGA001.1 )
  21. Gene Ruler 1 kb, GR 100 bp and GR 100 bp plus (Thermo Fisher Scientific, Thermo ScientificTM, catalog numbers: SM0311 , SM0241 and SM0321 , respectively)
  22. Dynabeads M-280 Streptavidin (Thermo Fisher Scientific, InvitrogenTM, catalog number: 11206D )
  23. Chelex 100 (Bio-Rad Laboratories, catalog number: 1421253 )
  24. Tris (BioShop, catalog number: TRS001.5 )
  25. 1 M Tris-HCl pH 8 (made of Tris)
  26. 1 M Tris-HCl pH 6.8 (as above)
  27. Acetic acid (Avantor Performance Materials, catalog number: BA8760114 )
  28. Sucrose (Sigma-Aldrich, catalog number: S0389 )
  29. Phenylmethylsulfonyl fluoride (Sigma-Aldrich, catalog number: P7626 )
  30. Formaldehyde (Sigma-Aldrich, catalog number: F8775-500ML or BioShop, catalog number: FOR201 )
  31. Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266 )
  32. Triton X-100 (Carl Roth, catalog number: 3051.3 )
  33. β-Mercaptoethanol (BioShop, catalog number: MER002 )
  34. Spermine (Sigma-Aldrich, catalog number: 85590-5G )
  35. 5 M NaCl (made of NaCl, Avantor Performance Materials, catalog number: 794121116 )
  36. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L3771 )
  37. Sodium bicarbonate (NaHCO3) (Sigma-Aldrich, catalog number: S5761 )
  38. Phenol (Sigma-Aldrich, catalog number: P4557 )
  39. Chloroform (Avantor Performance Materials, catalog number: BA6420113 )
  40. Isoamyl alcohol (Avantor Performance Materials, catalog number: 485560111 )
  41. Maxima Sybr Green/ROX qPCR Master Mix (2x) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: K0223 )
  42. Protease inhibitor cocktail (Sigma-Aldrich, catalog number: P8465 )
  43. 1x TE buffer (see Recipes)
  44. 1x TAE buffer (see Recipes)
  45. Cross-linking buffer (see Recipes)
  46. Honda buffer (see Recipes)
  47. TNE buffer (see Recipes)
  48. Extraction buffer (see Recipes)
  49. Pellet extraction buffer (see Recipes)
  50. Phenol/Chloroform/Isoamyl alcohol for DNA extraction (see Recipes)
  51. ChIP Dilution Buffer (see Recipes)
  52. Binding/Washing Buffer (see Recipes)


  1. Pipettes (Gilson, model: Pipetman® Neo or PZ HTL, model: Discovery Comfort )
  2. Fume hood
  3. Desiccator connected to the pump
  4. Mortars and pestles
  5. Small funnels
  6. Scale (KERN & SOHN, catalog number: EG 220-3NM )
  7. Rotator (Cole-Parmer, Stuart, model: SB3 )
  8. Centrifuges:
    Thermo Fisher Scientific, Thermo ScientificTM, model: HeraeusTM MultifugeTM X1R, catalog number: 75004250
    Thermo Fisher Scientific, Thermo ScientificTM, model: HeraeusTM BiofugeTM StratosTM Centrifuge, catalog number: 75005282
    Beckman Coulter, model: Allegra® X-30R Centrifuge, catalog number: A99471
    Eppendorf, model: MiniSpin® plus , catalog number: 0040262
    Gilson, model: CmC Lab CAPSULEFUGE PMC-880
  9. Thermomixer (Eppendorf, model: Thermomixer comfort , catalog number: 5355)
  10. Magnetic separator (Thermo Fisher Scientific, model: DynaMagTM-2, catalog number: 12321D )
  11. See Saw Rocker (Cole-Parmer, Stuart, model: SSL4 )
  12. Thermoblock (Grant Instruments, model: QBA2 )
  13. Vortex (Scientific Industries, model: Vortex-Genie 2 , catalog number: SI-0256)
  14. Systems for DNA electrophoresis (Mini-Sub Cell and Wide Mini-Sub Cell GT Complete Systems, Bio-Rad Laboratories, catalog numbers: 1640300 and 1640301 , respectively)
  15. Real-Time PCR System (Thermo Fisher Scientific, Applied BiosystemsTM, model: 7900HT Fast Real-Time PCR System, catalog number: 4329001 )


  1. 7900HT Fast Real-Time PCR System Software: SDSv2.4 (available on Thermo Scientific website)


  1. Cross-linking
    Cross-linking covalently preserves DNA-protein interactions for the further preparation steps. However, the excessive fixation may hinder later chromatin shearing and recognition of protein tags/epitopes. Use fresh formaldehyde, as its effective concentration drops over time, especially when exposed to air and light. Vacuum is used to increase tissue penetration. At the quenching step glycine becomes a substrate for the excess of formaldehyde.
    1. Collect 4 g of Arabidopsis thaliana rosette leaves into a 50 ml conical tube. If plants are dehydrated as in case of drought-treated material, 2 g of leaves can be enough to obtain similar efficiency of chromatin extraction. Cover leaves with a small piece of nylon mesh and close the tube with a riddled screw-cap.
    2. Add 37 ml of cross-linking buffer (see Recipes) (under the fume hood) and apply vacuum for 10 min at room temperature for cross-linking to achieve the pressure of 50 mBar. The plant tissue should look translucent after cross-linking.
    3. Stop cross-linking by adding 2.5 ml of 2 M glycine (to final conc. of 100 mM) and applying the vacuum infiltration for additional 5 min.

  2. Chromatin isolation and MNase digestion
    1. Wash plant tissues five times in sterile deionized water. Remove the water as much as possible by blotting the tissues between paper towels. This is important as ice crystals may destroy nuclei that would reduce efficiency of chromatin isolation. Freeze the tissue quickly in liquid nitrogen.
    2. Grind thoroughly the tissue to a very fine powder using pre-chilled mortars and pestles and ensure that the samples do not thaw during grinding.
    3. Transfer the plant powder into 50 ml conical tubes pre-cooled with liquid nitrogen. At this point you can store the material (for limited time) at -80 °C.
    4. Resuspend each sample in 20 ml of ice-cold Honda buffer (see Recipes).
    5. Vortex briefly (avoid floating) to mix and incubate on ice for 30 min with mixing on the rotator to homogenize the samples.
    6. Filter the homogenate through two layers of Miracloth and squeeze the Miracloth.
    7. Transfer the filtrate to a new 50 ml conical tube, and spin at 2,000 x g, 4 °C in a swing-out rotor (Heraeus) for 15 min.
    8. Resuspend the pellet very gently by pipetting in 20 ml of Honda buffer.
    9. Spin the suspension at 2,000 x g at 4 °C for 15 min.
    10. Repeat resuspension and centrifugation as above. There should be soft white pellet of nuclei forming at the bottom of the tube. If the pellet is still green, additional washes may be needed.
    11. Resuspend the pellet in 20 ml of Honda buffer without spermine.
    12. Spin the suspension at 2,000 x g at 4 °C for 8 min.
    13. Resuspend the nuclei pellet in 1 ml of ice-cold TNE buffer (see Recipes) and divide it into two 1.5 ml microcentrifuge tubes.
    14. In the presence of 4 mM CaCl2 (add 2 μl 1 M CaCl2 to each tube) add an appropriate number of MNase units (this needs to be established experimentally, we usually add between 8-20 U) to each tube and immediately place the tubes in a thermomixer pre-warmed to 37 °C. Incubate the tubes for 20 min with vigorous shaking (1,400 rpm) to liberate nucleosomes.
    15. Stop the reaction by placing the tubes on ice and adding 25 μl 0.5 M EGTA (to a final concentration of 25 mM).
    16. Spin at 14,000 x g in a microcentrifuge for 5 min at 4 °C.
    17. Save supernatant for further steps and store at -178 °C (liquid nitrogen). You can also save the pellet to check the efficiency of chromatin isolation and MNase digestion (the optional step [Step C3]).

  3. Detection of MNase digestion efficiency by DNA isolation (especially useful when optimizing MNase concentration)
    1. Reverse cross-linking and protein digestion
      Prior to DNA isolation, the crosslinks between DNA and proteins need to be reversed.
      1. Take 70 μl of chromatin isolate and add 400 μl of extraction buffer (see Recipes).
      2. Add 20 μl of 5 M NaCl and incubate at 65 °C for at least 4 h to overnight to reverse cross-linking.
      3. Add 10 μl of 0.5 M EDTA, 20 μl 1 M Tris-HCl pH 6.8, 1.7 μl RNase A (30 mg/ml) and incubate for 30 min at 37 °C.
      4. Add 1 μl Proteinase K (20 mg/ml) and incubate for 1.5 h at 45 °C to digest proteins.
    2. DNA precipitation
      1. Add an equal volume (500 μl) of phenol/chloroform/isoamyl alcohol and vortex briefly.
      2. Centrifuge at 14,000 x g for 5 min at RT and transfer the supernatant to a 2 ml Eppendorf tube.
      3. Add 1/10 volume of 3 M sodium acetate pH 5.2, 4 μl glycogen (20 mg/ml) and 2.5 volume of 100% EtOH, mix by inverting the tube several times and incubate at -80 °C for at least 40 min to precipitate the DNA.
      4. Centrifuge the sample at 14,000 x g for 15 min at 4 °C.
      5. Discard the supernatant, wash the pellet with 500 μl of cold 70% EtOH, centrifuge again for 5 min at 4 °C.
      6. Discard the supernatant and dry the pellet at RT. Dissolve the pellet in 20 μl and check on the 1.5% agarose gel (Figure 1). You should observe mainly mononucleosomal DNA fragments (of size ~150 bp). You can compare it to DNA isolated from the pellet after nuclei treatment with MNase (see optional step [Step C3] below).

        Figure 1. DNA isolated from MNase-treated chromatin. Lanes 1 to 4 correspond to different samples. DNA fragments of different length represent mono-, di- and trinucleosomal DNA (~150, ~300 and ~450 bp, respectively). The desired fragments are mononucleosomal, although overdigestion should be avoided as well.

    3. Optional: Isolation of DNA from pellet after MNase treatment
      Note: This protocol can be used to estimate how much genomic DNA remained in the pellet.
      1. Add 400 μl of Pellet extraction buffer (see Recipes) to the pellet obtained from MNase-treatment.
      2. Mix the tube vigorously for 15 min at 65 °C with 1,400 rpm shaking using thermomixer.
      3. Use 200 μl of the whole extract (no centrifugation) for incubation at 65 °C overnight to reverse crosslinking.
      4. Incubate with 1 μl RNase A (30 mg/ml) at 37 °C for 30 min.
      5. Add 1 μl of Proteinase K and incubate for 1.5 h at 45 °C.
      6. Add 200 μl phenol/chloroform/isoamyl alcohol (in a fume hood) to each sample and mix.
      7. Centrifuge at 16,000 x g for 5 min. Carefully transfer aqueous layer to fresh 1.5 ml tubes.
      8. Add 20 μl 3 M NaOAc to each tube, mix. Add 550 μl absolute ethanol, mix and incubate at -80 °C for at least 40 min.
      9. Centrifuge at 16,000 x g for 15 min at 4 °C to collect DNA.
      10. Wash pellet in 70% EtOH.
      11. Dry DNA pellet and resuspend in 50 μl TE buffer (see Recipes). Check 10 μl on the 1.5% gel along with a DNA ladder to estimate fragment size (Figure 2). You should observe that most of mono-, di-, and trinucleosomal DNA is present in the supernatant.

        Figure 2. Verification of chromatin MNase digestion. Lanes 1 and 3 contain DNA isolated from the supernatant (control and stress plants, respectively), lanes 2 and 4 contain DNA from the pellet (control and stress plants, respectively).

  4. Chromatin affinity purification (streptavidin to biotin)
    1. Take 25 µl of the Dynabeads M-280 Streptavidin slurry for each sample and put into the Maxymum Recovery (low retention) tube on ice.
    2. Wash three times each with 1 ml of ice-cold Binding/Washing Buffer (see Recipes). Collect the beads on tube wall on a magnetic separator (Figure 3).

      Figure 3. Separation of streptavidin beads (Dynabeads M-280) using a magnetic separator. Beads attached to one side of Maxymum Recovery tube wall, so the supernatant can be easily removed, and the beads could be washed.

    3. Resuspend the beads in 100 μl of cold Binding/Washing Buffer.
    4. Divide your chromatin supernatant (obtained in Step B17) into 140 μl portions (for ChAP) and 20 μl portions (for input).
    5. Dilute 140 μl of the chromatin supernatant ten times with ice-cold ChIP Dilution Buffer (add 1260 μl of ChIP Dilution Buffer) (with protease inhibitors added, see Recipes). Once you obtained a greater volume of the chromatin supernatant. Spin in a microcentrifuge at maximum speed for 10 min and then transfer to the tubes with beads (Step D3).
    6. Incubate overnight at 4 °C on a rotator such as Stuart SB3 Rotator with mild rotation (~15 rpm).
    7. Next morning, wash the beads on ice in the see-saw rocker:
      5 x 5 min Binding/Washing Buffer
      2 x 5 min TE
    8. After final wash in TE, before beads collection, transfer the samples to fresh Maxymum Recovery tubes to avoid releasing DNA bound to the tube walls.
    9. Add 100 µl thoroughly mixed 10% Chelex (w/w) to the beads in each tube. Add 200 µl 10% Chelex to 20 µl input portion. Chelex is a chelating resin used to protect DNA from degradation at high temperatures (Singer-Sam et al., 1989) with proven efficacy during the elution step in ChIP method (Nelson et al., 2005). Exact volumes of chromatin used for ChAP or input and volumes of their eluates are needed in calculations of ‘percent of input’.
    10. Boil the samples in a heating block at 99 °C for 10 min.
    11. Add 1 µl Proteinase K (20 mg/ml) to each tube and incubate at 43 °C for 1 h.
    12. Inactivate the Proteinase K by incubation at 99 °C for 10 min.
    13. Spin the samples for 1 min at maximum speed and transfer the supernatant to new low retention tubes.
    14. Use 2 µl of ChAP and input eluates in qPCR reactions.

Data analysis

  1. In ChAP-qPCR at least three biological replicates should be used for each experiment (three ChAP replicates and three corresponding input replicates). Each biological replicate should be repeated in qPCR in at least three technical repeats and the mean Ct should be used for further calculations.
  2. As MNase digestion results in DNA fragments of about 150 bp length, primers should be designed to produce short amplicons (perfectly about 80 bp). The quality of primers is absolutely critical for the accuracy of calculations. They should result in a near-to-perfect amplification of DNA (the amount of DNA in each cycle should increase of > 1.9 times). This should be verified by running qPCR reactions using serial dilutions of template DNA of known concentration. Based on this amplification the standard curve should be calculated (Figure 4). The slope of the standard curve should not deviate significantly from -3.33 (perfect amplification) and the R2 of the standard curve should be > 0.985 (1.000 is a perfect amplification). To verify whether the amplified DNA is indeed the expected product (e.g., not primer-dimers), analysis of the dissociation curve is recommended.
  3. Together with ChAP samples, the corresponding input samples need to be analyzed. In this case, a dilution should be adjusted to a level in which both ChAP and input samples do not differ in their Ct values by more than 3 cycles.
  4. Having successfully verified raw results of the qPCR run, percent of input (% of input) may be calculated as follows (Lin et al., 2012):
    % of input = 100 x 2ΔCt
    ΔCt = CtInput - log2(Input dilution factor) - CtChIP
  5. To easily compare results from differentially treated plants or even different experiments, normalization to the reference region is commonly used. In principle, it is a region in a genome in which the occupancy and distribution pattern of our protein of interest does not change irrespective of used conditions/treatments.
    Note: The example of such calculations is presented in the excel file.

    Figure 4. SDSv2.4 window printscreen. In the upper panel the standard curve plot calculated for a primer pair. The slope and R2 parameters inform about the PCR efficiency (ideal amplification has the slope close to -3.33 and R2 close to 1. In the bottom panel amplification plots for different DNA concentration. The software automatically sets the threshold and calculates Ct values.


  1. 1x TE buffer
    10 mM Tris-HCl (pH 8)
    1 mM EDTA
  2. 1x TAE buffer
    40 mM Tris
    20 mM acetic acid
    1 mM EDTA
  3. Cross-linking buffer
    0.4 M sucrose
    10 mM Tris-HCl (pH 8)
    1 mM EDTA
    1 mM PMSF
    1% formaldehyde
    Note: Make fresh buffer before each experiment. Presence of sucrose in the cross-linking buffer increases the efficiency of the DNA-protein cross-linking. The addition of the protease inhibitor (PMSF) should be done directly before using the buffer.
  4. Honda buffer
    25 mM Tris-HCl (pH 7.5)
    0.44 M sucrose
    10 mM MgCl2
    0.5% Triton X-100
    10 mM β-mercaptoethanol
    2 mM spermine
    1. Sterilize by filtering (and store at 8 °C) or make fresh before use.
    2. Add spermine and β-mercaptoethanol (70 μl per 100 ml) just before use.
    3. The last washing is performed without spermine.
  5. TNE buffer
    10 mM Tris-HCl (pH 8.0)
    100 mM NaCl
    1 mM EDTA
  6. Extraction buffer
    0.625% SDS
    0.125 M NaHCO3
  7. Pellet extraction buffer
    1x TE pH 8.0
    100 mM NaCl
    2% Triton X-100
    1% SDS
  8. Phenol/Chloroform/Isoamyl alcohol for DNA extraction
    25 parts of phenol (pH 8)
    24 parts of chloroform
    1 part of isoamyl alcohol
    Note: Prepare fresh before use, mix vigorously before each pipetting.
  9. ChIP Dilution Buffer
    16.7 mM Tris-HCl (pH 8)
    167 mM NaCl
    1.2 mM EDTA
    1.1% Triton X-100
    1 mM PMSF*
    Protease Inhibitor Cocktail*
  10. Binding/Washing Buffer
    20 mM Tris-HCl pH8
    150 mM NaCl
    2 mM EDTA
    1% Triton X-100
    0.1% SDS
    1 mM PMSF*

*Note: Add directly just before use.


This protocol was developed from the previously published paper (Sura et al., 2017), and is partially based on protocols published elsewhere (Saleh et al., 2008; Wierzbicki et al., 2008; Zilberman et al., 2008). This work was supported by grants from the National Science Centre grants (2016/21/B/NZ2/01757 to P.A.Z. and 2015/17/N/NZ1/00028 to W.S.) and Polish Ministry of Science and Higher Education grants (N/N303/313437 to P.A.Z., DI/2011/028641 to W.S.). The authors have no conflict of interest or competing interests to declare.


  1. de Boer, E., Rodriguez, P., Bonte, E., Krijgsveld, J., Katsantoni, E., Heck, A., Grosveld, F. and Strouboulis, J. (2003). Efficient biotinylation and single-step purification of tagged transcription factors in mammalian cells and transgenic mice. Proc Natl Acad Sci USA 100(13): 7480-5.
  2. Green, M. N. (1975). Avidin. Adv Protein Chem 29: 85-133.
  3. Lin, X., Tirichine, L. and Bowler, C. (2012). Protocol: Chromatin immunoprecipitation (ChIP) methodology to investigate histone modifications in two model diatom species. Plant Methods 8(1): 48.
  4. Mito, Y., Henikoff, J. G. and Henikoff, S. (2005). Genome-scale profiling of histone H3.3 replacement patterns. Nat Genet 37(10): 1090-1097.
  5. Nelson, J. D., Denisenko, O., Sova, P. and Bomsztyk, K. (2006). Fast chromatin immunoprecipitation assay. Nucleic Acids Res 34: 1-7.
  6. Saleh, A., Alvarez-Venegas, R. and Avramova, Z. (2008). An efficient chromatin immunoprecipitation (ChIP) protocol for studying histone modifications in Arabidopsis plants. Nat Protoc 3(6): 1018-1025.
  7. Singer-Sam, J., Tanguay, R. L. and Rjggs, A. O. (1989). Use of Chelex to improve PCR signal from a small number of cells. Amplifications: A Forum for PCR Users: 11.
  8. Sura, W., Kabza, M., Karlowski, W. M., Bieluszewski, T., Kus-Slowinska, M., Paweloszek, L., Sadowski, J. and Ziolkowski, P. A. (2017). Dual role of the histone variant H2A.Z in transcriptional regulation of stress-response genes. Plant Cell 29(4): 791-807.
  9. Wierzbicki, A. T., Haag, J. R. and Pikaard, C. S. (2008). Noncoding transcription by RNA polymerase Pol IVb/Pol V mediates transcriptional silencing of overlapping and adjacent genes. Cell 135(4): 635-648.
  10. Zentner, G. E. and Henikoff, S. (2014). High-resolution digital profiling of the epigenome. Nat Rev Genet 15(12): 814-827.
  11. Zilberman, D., Coleman-Derr, D., Ballinger, T. and Henikoff, S. (2008). Histone H2A.Z and DNA methylation are mutually antagonistic chromatin marks. Nature 456 (7218): 125-129.


染色质亲和纯化(ChAP)被广泛用于研究染色质结构和与DNA相互作用的蛋白质复合物。 在这里,我们提出了一种有效的从拟南芥莲座叶中ChAP的方法,其中使用了体内生物素化系统。 染色质被Micrococcal核酸酶(MNase)消化,因此核小体的分布也被实现。 体内生物素化系统最初是为黑腹果蝇而开发的(Mito et al。2005),但是所提出的方案是专门为 拟南芥(Sura et。,2017)。

【背景】染色质免疫沉淀(ChIP)成为研究染色质结构和组织的最重要和最常用的技术之一。但是,它需要高质量的抗体,不会与非特异性靶标发生交叉反应。在含有细胞壁并富含光合作用相关化合物和蛋白质的植物中,这是相当难以实现的,这些化合物和蛋白质经常引起交叉反应性问题。另一方面,获得稳定的转基因生物是植物常规和容易的策略。由于这些原因,大多数植物研究人员选择基因标签,获得融合蛋白,并用ChIP替代方法即染色质亲和纯化(ChAP)来研究染色质。 ChAP技术已被证明在植物染色质研究中非常有效(Zentner和Henikoff,2014)。此外,它通常比经典ChIP便宜,因为它不需要产生抗体,并且通常比ChIP更有效,因为标签以比直接针对感兴趣的蛋白质产生的抗体更高的亲和力被识别。 ChAP的一个缺点是它不能用于研究翻译后组蛋白修饰。在所提出的方案中,蛋白质用短的生物素连接酶识别肽(BLRP)标记,其通过大肠杆菌BirA生物素连接酶(de Boer >等人,2003)。因此,使用基于链霉抗生物素蛋白的纯化系统(例如,Dynabeads M-280链霉抗生物素蛋白,Invitrogen)来纯化标记的蛋白质。由于链霉亲和素对生物素具有非常高的亲和力(解离常数约为10 -14 mol / L),因此生物素与链霉抗生物素蛋白的结合是自然界已知的最强非共价相互作用之一( Green,1975)。或者,如果使用适合的最终纯化系统,则所提供的方案可成功地应用于用其他标签(例如,MYC,GFP,HA,FLAG)标记的蛋白质的ChAP。

关键字:染色质亲和纯化, ChAP-qPCR, 染色质免疫沉淀, ChIP, 染色质MNase消化, 核小体占位, 组蛋白分布, 拟南芥


  1. 标准移液器吸头
  2. 15毫升和50毫升锥形离心管
  3. 尼龙网(孔径80μm,膜解决方案,目录号:MENY090080)
  4. 纸巾
  5. Miracloth(Merck,Calbiochem,目录号:475855)
  6. 1.5-2.0毫升微量离心管
  7. 2ml和1.5ml低保留管(Maxymum回收微管; Corning,Axygen,产品目录号:MCT-200-L-C和MCT-150-L-C)
  8. 植物材料:A。表达E的拟南芥植物。在具有与生物素连接酶识别肽(BLRP)融合的感兴趣的基因的强启动子(例如,拟南芥Act8)的控制下的大肠杆菌BirA生物素连接酶( de Boer等人,2003; Mito et al。,2005)
    注:植物可能处于从一周龄幼苗到成年的不同发育阶段,开花前。一般来说,年轻的材料,染色质分离的效率更高。而且,年轻的植物提供用于染色质提取的组织更高的同质性和重复之间的更小的变异。 BirA生物素连接酶可以从不同的启动子表达,但是表达应保持在高水平。其他可能的启动子包括遍在蛋白10启动子(UBQ10)和花椰菜镰孢病毒(CaMV)35S启动子。可诱导的系统,例如地塞米松(DEX) - 可诱导的,也可以使用,然而这在我们的实验室中未被测试。
  9. 2M甘氨酸(由甘氨酸制造,Sigma-Aldrich,目录号:33226)
  10. 液氮,冰
  11. 1M CaCl 2(由CaCl 2,Sigma-Aldrich制造,目录号:C5670)。
  12. 微球菌核酸酶(Thermo Fisher Scientific,Thermo Scientific TM,目录号:EN0181)
  13. 0.5 M EGTA(由EGTA,BioShop制造,目录编号:EGT101)
  14. 0.5M EDTA(由EDTA制造,BioShop,目录号:EDT001)
  15. RNase A(Sigma-Aldrich,目录号:R4642)
  16. 蛋白酶K(Thermo Fisher Scientific,Thermo Scientific TM,目录号:EO0491)
  17. 3M醋酸钠pH 5.2(由CH 3 COONa,INC Biomedicals Inc.制造)
  18. 糖原(Thermo Fisher Scientific,Thermo Scientific TM,目录号:R0561)
  19. 乙醇(Avantor性能材料,目录号:BA6420113)
  20. 琼脂糖(BioShop,目录号:AGA001.1)
  21. 基因标记1kb,GR 100bp和GR 100bp加(Thermo Fisher Scientific,Thermo Scientific TM,目录号分别为SM0311,SM0241和SM0321)。
  22. Dynabeads M-280抗生蛋白链菌素(Thermo Fisher Scientific,Invitrogen TM,目录号:11206D)
  23. Chelex 100(Bio-Rad Laboratories,目录号:1421253)
  24. Tris(BioShop,目录号:TRS001.5)
  25. 1M Tris-HCl pH 8(由Tris制成)
  26. 1M Tris-HCl pH 6.8(如上)
  27. 乙酸(Avantor高性能材料,目录号:BA8760114)
  28. 蔗糖(Sigma-Aldrich,目录号:S0389)
  29. 苯基甲基磺酰氟(Sigma-Aldrich,目录号:P7626)
  30. 甲醛(Sigma-Aldrich,目录号:F8775-500ML或BioShop,目录号:FOR201)
  31. 氯化镁(MgCl 2)(Sigma-Aldrich,目录号:M8266)
  32. Triton X-100(Carl Roth,目录号:3051.3)
  33. β-巯基乙醇(BioShop,目录号:MER002)
  34. 精胺(Sigma-Aldrich,目录号:85590-5G)
  35. 5M NaCl(由NaCl制成,Avantor Performance Materials,目录号:794121116)
  36. 十二烷基硫酸钠(SDS)(Sigma-Aldrich,目录号:L3771)
  37. 碳酸氢钠(NaHCO 3)(Sigma-Aldrich,目录号:S5761)
  38. 苯酚(Sigma-Aldrich,目录号:P4557)
  39. 氯仿(Avantor高性能材料,目录号:BA6420113)
  40. 异戊醇(Avantor高性能材料,目录号:485560111)
  41. Maxima Sybr Green / ROX qPCR Master Mix(2x)(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:K0223)
  42. 蛋白酶抑制剂混合物(Sigma-Aldrich,目录号:P8465)
  43. 1倍的TE缓冲液(见食谱)
  44. 1倍TAE缓冲液(见食谱)
  45. 交叉缓冲区(见食谱)
  46. 本田缓冲区(见食谱)
  47. TNE缓冲液(见食谱)
  48. 提取缓冲液(见食谱)
  49. 颗粒提取缓冲液(见食谱)
  50. 苯酚/氯仿/异戊醇DNA提取(见食谱)
  51. ChIP稀释缓冲液(见食谱)
  52. 装订/洗涤缓冲液(见食谱)


  1. 移液器(Gilson,型号:Pipetman Neo或PZ HTL,型号:Discovery Comfort)
  2. 通风柜
  3. 干燥器连接到泵
  4. 迫击炮和杵
  5. 小漏斗
  6. 规模(KERN& SOHN,目录号:EG 220-3NM)
  7. 旋转器(Cole-Parmer,Stuart,型号:SB3)
  8. 离心机:
    赛默飞世尔科技Thermo Scientific TM型号:Heraeus TM Multifuge TM X1R,产品目录号:75004250
    Thermo Fisher Scientific Thermo Scientific TM型号:Heraeus TM Biofuge TM Stratos TM离心机,目录号:75005282
    Beckman Coulter型号:Allegra X-30R离心机,目录号:A99471
    Eppendorf,型号:MiniSpin ®,产品目录号:0040262
    Gilson,模型:CMC实验室CAPSULEFUGE PMC-880
  9. Thermomixer(Eppendorf,型号:Thermomixer comfort,目录编号:5355)
  10. 磁性分离器(Thermo Fisher Scientific,型号:DynaMag TM -2,目录号:12321D)
  11. 见锯机(Cole-Parmer,Stuart,型号:SSL4)
  12. Thermoblock(Grant Instruments,型号:QBA2)
  13. 涡流(Scientific Industries,型号:Vortex-Genie 2,产品目录号:SI-0256)
  14. 用于DNA电泳的系统(Mini-Sub Cell和Wide Mini-Sub Cell GT Complete Systems,Bio-Rad Laboratories,产品目录号分别为1640300和1640301)
  15. 实时PCR系统(Thermo Fisher Scientific,Applied Biosystems TM,型号:7900HT Fast Real-Time PCR System,目录号:4329001)


  1. 7900HT快速实时PCR系统软件:SDSv2.4(可在Thermo Scientific网站上获得)


  1. 交叉链接
    1. 收集4克拟南芥叶莲花叶片到50毫升锥形管中。如果植物在干旱处理物质的情况下脱水,则2g的叶子可以获得类似的染色质提取效率。用一小片尼龙网盖住盖子,用螺丝帽盖住管子。
    2. 加入37 ml交联缓冲液(见配方)(在通风橱下),室温下真空10 min交联以达到50 mBar的压力。
    3. 停止交联加入2.5毫升2 M甘氨酸(最终浓度为100毫米),并应用真空渗透额外5分钟。

  2. 染色质分离和MNase消化
    1. 用无菌去离子水清洗植物组织5次。通过吸干纸巾之间的组织,尽可能地去除水分。这一点很重要,因为冰晶可能会破坏会降低染色质分离效率的细胞核。
    2. 使用预先冷却的研钵和研杵将组织彻底研磨成细粉,确保样品在研磨过程中不会解冻。
    3. 将植物粉末转移到用液氮预冷却的50ml锥形管中。此时,您可以将材料(限定时间)储存在-80°C。
    4. 将每个样品重新悬浮在20毫升冰冷的本田缓冲液中(见食谱)。
    5. 短暂涡旋(避免浮动)混合并在冰上孵育30分钟,同时在旋转器上混合以均化样品。
    6. 通过两层Miracloth过滤匀浆,挤压Miracloth。
    7. 将滤液转移到新的50ml锥形管中,并在摆动转子(Heraeus)中在2000℃,4℃旋转15分钟。

    8. 在20毫升本田缓冲液中移液,非常轻柔地重悬沉淀

    9. 在4℃下将悬浮液在2000×g下旋转15分钟
    10. 重复如上重悬和离心。在管的底部应该形成核白色的软白色颗粒。如果颗粒仍然是绿色,可能需要额外的清洗。

    11. 在20毫升不含精胺的本田缓冲液中重悬沉淀

    12. 在4℃下将悬浮液旋转2,000 x g。8分钟
    13. 在1毫升冰冷的TNE缓冲液中重悬细胞核(参见食谱),并将其分成两个1.5毫升微量离心管。
    14. 在4mM CaCl 2(向每个管中加入2μl1M CaCl 2)存在下加入适量的MNase单位(这需要通过实验建立,我们通常在每个试管中加入8-20U)并立即将试管置于预热至37℃的热混合器中。
    15. 将试管置于冰上并加入25μl0.5M EGTA(终浓度为25mM),停止反应。

    16. 在微量离心机中于14℃旋转14,000×g 5分钟。
    17. 保存上清液进一步的步骤,并储存在-178°C(液氮)。您也可以保存颗粒来检查染色质分离和MNase消化(可选步骤[步骤C3])的效率。

  3. 通过DNA分离检测MNase消化效率(尤其在优化MNase浓度时有用)
    1. 反向交联和蛋白质消化
      1. 取70μl染色质分离物并加入400μl提取缓冲液(见食谱)。
      2. 加入20μL5MNaCl,并在65°C孵育至少4小时至过夜以逆转交联。
      3. 加入10μl的0.5M EDTA,20μl1M Tris-HCl pH6.8,1.7μlRNA酶A(30mg / ml)并在37℃孵育30分钟。
      4. 加入1微升蛋白酶K(20毫克/毫升)孵育1.5小时,在45°C消化蛋白质。
    2. DNA沉淀
      1. 加入等体积的(500μl)苯酚/氯仿/异戊醇并短暂涡旋。
      2. 在室温下14,000×g离心5分钟并将上清液转移到2ml Eppendorf管中。
      3. 加入1/10体积的pH5.2的3M乙酸钠,4μl糖原(20mg / ml)和2.5体积的100%EtOH,通过颠倒管混合几次并在-80℃下孵育至少40分钟至沉淀DNA。

      4. 在14,000×g的条件下离心15分钟
      5. 弃去上清,用500μl冷的70%乙醇洗涤沉淀,在4℃下再次离心5分钟。
      6. 弃去上清液并在室温下干燥沉淀。将沉淀溶解在20μl中,并在1.5%琼脂糖凝胶上检查(图1)。您应该主要观察单核细胞DNA片段(大小约150 bp)。您可以将其与从MNase核处理后的沉淀中分离出的DNA进行比较(参见下面的可选步骤[步骤C3])。


    3. 可选:在MNase处理后从沉淀中分离DNA

      1. 加入400μlPellet提取缓冲液(见配方)

      2. 。在65°C用力混合器搅拌15分钟
      3. 使用200μl的全部提取物(无离心)在65℃孵育过夜,以逆转交联。
      4. 用1μlRNase A(30 mg / ml)37°C孵育30分钟。
      5. 加入1微升蛋白酶K,并在45°C孵育1.5小时。
      6. 向每个样品中加入200μl苯酚/氯仿/异戊醇(在通风橱中)并混合。
      7. 16,000×g离心5分钟。小心转移水层到新鲜的1.5毫升管。
      8. 加入20μL3M NaOAc到每个管,混合。加入550μl无水乙醇,混合并在-80℃下孵育至少40分钟。
      9. 在4℃下以16,000×gg离心15分钟以收集DNA。
      10. 用70%乙醇洗沉淀。
      11. 干燥DNA沉淀并重悬于50μlTE缓冲液中(参见食谱)。在1.5%凝胶上检测10μl以及DNA梯度以估计片段大小(图2)。你应该观察到,大部分单核,双核和三核DNA都存在于上清液中。


  4. 染色质亲和纯化(链霉亲和素与生物素)
    1. 每个样品取25μl的Dynabeads M-280 Streptavidin浆液,放入冰上的Maxymum Recovery(低保留)管中。
    2. 每次用1ml冰冷的Binding / Washing Buffer洗涤三次(见食谱)。将磁珠收集在磁选机的管壁上(图3)。

      图3.使用磁力分离器分离链霉抗生物素蛋白珠(Dynabeads M-280)。 珠附着在Maxymum Recovery管壁的一侧,因此上清液可以很容易地被去除,珠子可以被清洗。

    3. 在100μl冷结合/洗涤缓冲液中重悬珠。
    4. 将染色质上清液(步骤B17获得)分成140μl(ChAP)和20μl(用于输入)。
    5. 用冰冷的ChIP稀释缓冲液(加入1260μlChIP稀释缓冲液)(加入蛋白酶抑制剂,参见食谱)稀释140μl染色质上清液10次。一旦获得更大量的染色质上清液。在微型离心机中以最大速度旋转10分钟,然后转移到具有珠子的管(步骤D3)。

    6. 温和旋转(〜15 rpm),在斯图尔特SB3旋转器等旋转器上于4°C孵育过夜。
    7. 第二天早上,在跷跷板摇摆器上用冰块清洗珠子。
      5 x 5分钟结合/清洗缓冲液
    8. 在TE中进行最后的清洗之后,在收集珠之前,将样品转移到新鲜的Maxymum回收管中以避免释放结合到管壁的DNA。
    9. 将100μl彻底混合的10%Chelex(w / w)加入每个管中的珠子中。加入200μl10%Chelex至20μl输入部分。 Chelex是用于在高温下保护DNA免受降解的螯合树脂(Singer-Sam等人,1989),其在ChIP方法的洗脱步骤中具有证实的功效(Nelson等人, 2005年)。在计算“输入百分比”时,需要用于ChAP或输入的精确体积的染色质和其洗脱液的体积。
    10. 将样品在99℃的加热块中煮沸10分钟。

    11. 加入1μl蛋白酶K(20 mg / ml)到每个管中,并在43°C孵育1小时。
    12. 通过在99℃温育10分钟灭活蛋白酶K.
    13. 将样品以最大速度旋转1分钟,并将上清液转移到新的低保留管中。
    14. 使用2μl的ChAP,并在qPCR反应中输入洗脱液。


  1. 在ChAP-qPCR中,每个实验至少应使用三个生物学重复(三个ChAP重复和三个相应的重复输入)。每个生物学重复应在qPCR中重复至少三次技术重复,平均Ct应用于进一步计算。
  2. 由于MNase消化产生约150bp长度的DNA片段,所以应设计引物以产生短的扩增子(完全约80bp)。引物的质量对于计算的准确性是绝对关键的。它们应该导致DNA的接近完美的扩增(每个循环中DNA的量应该增加> 1.9倍)。这应该通过使用已知浓度的模板DNA的连续稀释进行qPCR反应来验证。基于这种扩增,应计算标准曲线(图4)。标准曲线的斜率应该不偏离-3.33(完全放大),并且标准曲线的R2应当> 0.985(1.000是一个完美的放大)。为了验证扩增的DNA是否确实是预期的产物(例如,而不是引物二聚体),推荐分析解离曲线。
  3. 与ChAP样本一起,需要分析相应的输入样本。在这种情况下,应将稀释度调整到ChAP和输入样品的Ct值相差不超过3个循环的水平。
  4. 在成功验证了qPCR运行的原始结果之后,输入百分比(输入百分比)可以如下计算(Lin等人,2012):

    输入的百分比= 100 x 2 <ΔCt


    ΔCt= Ct <输入> -log <2>(输入稀释因子) - Ct
  5. 为了容易地比较来自差异处理植物或甚至不同实验的结果,通常使用归一化到参考区域。原则上,它是一个基因组中的一个区域,其中感兴趣的蛋白质的占用和分布模式不会改变,而不考虑所使用的条件/处理。
    注意:这种计算的例子在 excel文件

    图4. SDSv2.4窗口printscreen 在上方面板中,为引物对计算的标准曲线图。斜率和R2参数提示PCR效率(理想扩增的斜率接近于-3.33,R2接近于1.在不同DNA浓度的底部扩增曲线图中,软件自动设置阈值并计算Ct值。


  1. 1x TE缓冲液
    10 mM Tris-HCl(pH 8)
    1 mM EDTA
  2. 1X TAE缓冲区
    40 mM Tris
    1 mM EDTA
  3. 交叉缓冲
    0.4 M蔗糖
    10 mM Tris-HCl(pH 8)
    1 mM EDTA
  4. 本田缓冲区
    25 mM Tris-HCl(pH 7.5)
    0.44 M蔗糖
    10mM MgCl 2 / sup>
    0.5%Triton X-100
    10 mMβ-巯基乙醇
    1. 通过过滤消毒(并在8°C储存)或使用前新鲜。
    2. 在使用前加入精胺和β-巯基乙醇(每100毫升70微升)。
    3. 最后一次清洗是在没有精胺的情况下进行的。
  5. TNE缓冲区
    10 mM Tris-HCl(pH 8.0)
    100 mM NaCl
    1 mM EDTA
  6. 提取缓冲区
    0.125M NaHCO 3 3
  7. 颗粒提取缓冲液
    1x TE pH 8.0
    100 mM NaCl
    2%Triton X-100
  8. 苯酚/氯仿/异戊醇DNA提取
    25份苯酚(pH 8)
  9. 芯片稀释缓冲液
    16.7mM Tris-HCl(pH8)
    167 mM NaCl
    1.2 mM EDTA
    1.1%Triton X-100
    1 mM PMSF *
  10. 装订/清洗缓冲液
    20mM Tris-HCl pH8
    150 mM NaCl
    2 mM EDTA
    1%Triton X-100
    1 mM PMSF *



该协议是从先前发表的论文(Sura等人,2017年)开发的,部分基于在别处公布的协议(Saleh等人,2008; Wierzbicki,等人,2008; Zilberman等人,2008)。这项工作得到了国家科学中心赠款(2016/21 / B / NZ2 / 01757到PAZ和2015/17 / N / NZ1 / 00028到WS)和波兰科学和高等教育部拨款(N / N303 / 313437至PAZ,DI / 2011/028641至WS)。作者没有利益冲突或竞争利益申报。


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引用:Sura, W. and Ziolkowski, P. A. (2018). Chromatin Affinity Purification (ChAP) from Arabidopsis thaliana Rosette Leaves Using in vivo Biotinylation System. Bio-protocol 8(1): e2677. DOI: 10.21769/BioProtoc.2677.