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Chromatin IP

Required Reagents

Reagents Included:

  1. Glycine Solution (10X)
  2. Buffer A (4X)
  3. Buffer B (4X)
  4. ChIP Buffer (10X)
  5. ChIP Elution Buffer (2X)
  6. 5 M NaCl
  7. 0.5 M EDTA
  8. 1M DTT
  9. DNA Binding Reagent A (add 12 ml isopropanol before use)
  10. DNA Wash Reagent B (add 24 ml ethanol before use)
  11. DNA Elution Reagent C
  12. DNA Spin Columns
  13. Protease Inhibitor Cocktail (200X)
  14. RNAse A (10 mg/ml)
  15. Micrococcal Nuclease (2000 gel units/µl)
  16. Proteinase K (20 mg/ml)
  17. SimpleChIP® Human RPL30 Exon 3 Primers #7014
  18. SimpleChIP® Mouse RPL30 Intron 2 Primers #7015
  19. Histone H3 (D2B12) XP® Rabbit mAb (ChIP Formulated) #4620
  20. Normal Rabbit IgG #2729
  21. ChIP-Grade Protein G Agarose Beads #9007

Reagents Not Included:

  1. Formaldehyde (37%)
  2. Ethanol (96-100%)
  3. Isopropanol
  4. 1X PBS
  5. Nuclease-free water
  6. Taq DNA polymerase
  7. dNTP Mix

I. Tissue Cross-linking and Sample Preparation:

When harvesting tissue, remove unwanted material such as fat and necrotic material from the sample. Tissue can then be processed and cross-linked immediately, or frozen on dry ice for processing later. For optimal chromatin yield and ChIP results, use 25 mg of tissue for each immunoprecipitation to be performed. The chromatin yield does vary between tissue types and some tissues may require more than 25 mg for each immunoprecipitation. Please see Appendix A for more information regarding the expected chromatin yield for different types of tissue. One additional chromatin sample should be processed for Analysis of Chromatin Digestion and Concentration (Section IV).

Before starting:

  • Remove and warm 200X Protease Inhibitor Cocktail (PIC) and 10X Glycine Solution. Make sure PIC is completely thawed.
  • Prepare 3 ml of Phosphate Buffered Saline (PBS) + 15 μl 200X PIC per 25 mg of tissue to be processed and place on ice.
  • Prepare 45 μl of 37% formaldehyde per 25 mg of tissue to be processed and keep at room temperature. Use fresh formaldehyde that is not past the manufacturer’s expiration date.

A. Cross-linking

  1. Weigh the fresh or frozen tissue sample. Use 25 mg of tissue for each IP to be performed.
  2. Place tissue sample in a 60 mm or 100 mm dish and finely mince using a clean scalpel or razor blade. Keep dish on ice. It is important to keep the tissue cold to avoid protein degradation.
  3. Transfer minced tissue to a 15 ml conical tube.
  4. Add 1 ml of PBS + PIC per 25 mg tissue to the conical tube.
  5. To crosslink proteins to DNA, add 45 μl of 37% formaldehyde per 1 ml of PBS + PIC and rock at room temp for 20 min. Final formaldehyde concentration is 1.5%.
  6. Stop cross-linking by adding 100 μl of 10x glycine per 1 ml of PBS + PIC and mix for 5 min at room temperature.
  7. Centrifuge tissue at 1,500 rpm in a bench top centrifuge for 5 min at 4°C.
  8. Remove supernatant and wash one time with 1 ml PBS + PIC per 25 mg tissue.
  9. Repeat centrifugation at 1,500 rpm in a bench top centrifuge for 5 min at 4°C.
  10. Remove supernatant and resuspend tissue in 1 ml PBS + PIC per 25 mg tissue and store on ice. Disaggregate tissue into single-cell suspension using a Medimachine (Part B) or Dounce homogenizer (Part C).

B. Tissue Disaggregation Using Medimachine from BD Biosciences (part #340587)

  1. Cut off the end of a 1000 μL pipette tip to enlarge the opening for transfer of tissue chunks.
  2. Transfer 1 ml of tissue resuspended in PBS + PIC into the top chamber of a 50 mm medicone (part #340592).
  3. Grind tissue for 2 min according to manufacturer’s instructions.
  4. Collect cell suspension from the bottom chamber of the medicone using a 1 ml syringe and 18 gauge blunt needle. Transfer cell suspension to a 15 ml conical tube and place on ice.
  5. Repeat steps 2 to 4 until all the tissue is processed into a homogenous suspension.
  6. If more grinding is necessary, add more PBS + PIC to tissue. Repeat steps 2 to 5 until all tissue is ground into a homogeneous suspension.
  7. Check for single-cell suspension by microscope (optional).
  8. Centrifuge cells at 1,500 rpm in a bench top centrifuge for 5 min at 4°C.
  9. Remove supernatant from cells and immediately continue with Nuclei Preparation and Chromatin Digestion (Section III).

C. Tissue Disaggregation Using a Dounce Homogenizer:

  1. Transfer tissue resuspended in PBS + PIC to a Dounce homogenizer.
  2. Disaggregate tissue pieces with 20-25 strokes. Check for single-cell suspension by microscope (optional).
  3. Transfer cell suspension to a 15 ml conical tube and centrifuge at 1,500 rpm in a bench top centrifuge for 5 min at 4°C.
  4. Remove supernatant from cells and immediately continue with Nuclei Preparation and Chromatin Digestion (Section III).

II. Cell Culture Cross-linking and Sample Preparation:

For optimal ChIP results, use approximately 4 X 106 cells for each immunoprecipitation to be performed. For HeLa cells, this is equivalent to half of a 15 cm culture dish containing cells that are 90% confluent in 20 ml of growth medium. One additional sample should be processed for Analysis of Chromatin Digestion and Concentration (Section IV). Include one extra dish of cells in experiment to be used for determination of cell number using a hemocytometer.

Before starting:

  • Remove and warm 200X Protease Inhibitor Cocktail (PIC) and 10X Glycine Solution. Make sure PIC is completely thawed.
  • Prepare 2 ml of Phosphate Buffered Saline (PBS) + 10 μl 200X PIC per 15 cm dish to be processed and place on ice.
  • Prepare 40 ml of PBS per 15 cm dish to be processed and place on ice.
  • Prepare 540 μl of 37% formaldehyde per 15 cm dish of cells to be processed and keep at room temperature. Use fresh formaldehyde that is not past the manufacturer’s expiration date.
  1. To crosslink proteins to DNA, add 540 μl of 37% formaldehyde to each 15 cm culture dish containing 20 ml medium. Swirl briefly to mix and incubate 10 min at room temperature. Final formaldehyde concentration is 1%. Addition of formaldehyde may result in a color change of the medium.
  2. Add 2 ml of 10X glycine to each 15 cm dish containing 20 ml medium, swirl briefly to mix, and incubate 5 min at room temperature. Addition of glycine may result in a color change of the medium.
  3. For suspension cells, transfer cells to a 50 ml conical tube, centrifuge at 1,500 rpm in a bench top centrifuge 5 min at 4°C and wash pellet two times with 20 ml ice-cold PBS. Remove supernatant and immediately continue with Nuclei Preparation and Chromatin Digestion (Section III).
  4. For adherent cells, remove media and wash cells two times with 20 ml ice-cold 1X PBS, completely removing wash from culture dish each time.
  5. Add 2 ml ice-cold PBS + PIC to each 15 cm dish. Scrape cells into cold buffer. Combine cells from all culture dishes into one 15 ml conical tube.
  6. Centrifuge cells at 1,500 rpm in a bench top centrifuge for 5 min at 4°C. Remove supernatant and immediately continue with Nuclei Preparation and Chromatin Digestion (Section III).

III. Nuclei Preparation and Chromatin Digestion:

One immunoprecipitation preparation (IP prep) is defined as 25 mg of disaggregated tissue or 4 x 106 tissue culture cells.

Before starting:

  • Remove and warm 200X Protease Inhibitor Cocktail (PIC) and 1 M DTT. Make sure both are completely thawed and DTT crystals are completely in solution.
  • Remove and warm 10X ChIP Buffer and ensure SDS is completely in solution.
  • Prepare 1 ml 1X Buffer A (250 μl 4X Buffer A + 750 μl water) + 0.5 μl 1M DTT + 5 μl 200X PIC per IP prep and place on ice.
  • Prepare 1.1 ml 1X Buffer B (275 μl 4X Buffer B + 825 μl water) + 0.55 μl 1M DTT per IP prep and place on ice.
  • Prepare 100 μl 1X ChIP Buffer (10 μl 10X ChIP Buffer + 90 μl water) + 0.5 μl 200X PIC per IP prep and place on ice.
  1. Resuspend cells in 1 ml ice-cold Buffer A + DTT + PIC per IP prep. Incubate on ice for 10 min. Mix by inverting tube every 3 min.
  2. Pellet nuclei by centrifugation at 3,000 rpm in a bench top centrifuge for 5 min at 4°C. Remove supernatant and resuspend pellet in 1 ml ice-cold Buffer B + DTT per IP prep. Repeat centrifugation, remove supernatant, and resuspend pellet in 100 μl Buffer B + DTT per IP prep. Transfer sample to a 1.5 ml microcentrifuge tube, up to 1 ml total per tube.
  3. Add 0.5 μl of Micrococcal Nuclease per IP prep, mix by inverting tube several times and incubate for 20 min at 37°C with frequent mixing to digest DNA to length of approximately 150-900 bp. Mix by inversion every 3 to 5 min. The amount of Micrococcal Nuclease required to digest DNA to the optimal length may need to be determined empirically for individual tissues and cell lines (see Appendix B). HeLa nuclei digested with 0.5 μl Micrococcal Nuclease per 4 x 106 cells and mouse liver tissue digested with 0.5 μl Micrococcal Nuclease per 25 mg of tissue gave the appropriate length DNA fragments (see Figure 3).
  4. Stop digest by adding 10 μl of 0.5 M EDTA per IP prep and placing tube on ice.
  5. Pellet nuclei by centrifugation at 13,000 rpm in a microcentrifuge for 1 min at 4°C and remove supernatant.
  6. Resuspend nuclear pellet in 100 μl of 1X ChIP buffer + PIC per IP prep and incubate on ice for 10 min.
  7. Sonicate up to 500 μl of lysate per 1.5 ml microcentrifuge tube with several pulses to break nuclear membrane. Incubate samples for 30 sec on wet ice between pulses. Optimal conditions required for complete lysis of nuclei can be determined by observing nuclei under light microscope before and after sonication. HeLa nuclei were completely lysed after 3 sets of 20-sec pulses using a VirTis Virsonic 100 Ultrasonic Homogenizer/Sonicator at setting 6 with a 1/8-inch probe. Alternatively, nuclei can be lysed by homogenizing the lysate 20 times in a Dounce homogenizer; however, lysis may not be as complete.
  8. Clarify lysates by centrifugation at 10,000 rpm in a microcentrifuge for 10 min at 4°C.
  9. Transfer supernatant to a new tube. This is the cross-linked chromatin preparation, which should be stored at -80°C until further use. Remove 50 μl of the chromatin preparation for Analysis of Chromatin Digestion and Concentration (Section IV).

IV. Analysis of Chromatin Digestion and Concentration (Recommended Step)

  1. To the 50 μl chromatin sample (from Step 9 in Section III), add 100 μl nuclease-free water, 6 μl 5 M NaCl, and 2 μl RNAse A. Vortex to mix and incubate samples at 37°C for 30 min.
  2. To each RNAse A-digested sample, add 2 μl Proteinase K. Vortex to mix and incubate samples at 65°C for 2 h.
  3. Purify DNA from samples using DNA purification spin columns as described in Section VII.
  4. After purification of DNA, remove a 10 μl sample and determine DNA fragment size by electrophoresis on a 1% agarose gel with a 100 bp DNA marker. DNA should be digested to a length of approximately 150-900 bp (1 to 5 nucleosomes; see Figure 3).
  5. To determine DNA concentration, transfer 2 μl of purified DNA to 98 μl nuclease-free water to give a 50-fold dilution and read the OD260. The concentration of DNA in μg/ml is OD260 x 2,500. DNA concentration should ideally be between 50 and 200 μg/ml.

NOTE: For optimal ChIP results, it is highly critical that the chromatin is of appropriate size and concentration. Over-digestion of chromatin may diminish signal in the PCR quantification. Under-digestion of chromatin may lead to increased background signal and lower resolution. Adding too little chromatin to the IP may result in diminished signal in the PCR quantification. A protocol for optimization of chromatin digestion can be found in Appendix B.

V. Chromatin Immunoprecipitation:

For optimal ChIP results, use approximately 5 to 10 μg of digested, cross-linked chromatin (as determined in Section IV) per immunoprecipitation. This should be roughly equivalent to a single 100 μl IP prep from 25 mg of disaggregated tissue or 4 x 106 tissue culture cells. Typically, 100 μl of digested chromatin is diluted into 400 μl 1X ChIP Buffer prior to the addition of antibodies. However, if more than 100 μl of chromatin is required per IP, the cross-linked chromatin preparation does not need to be diluted as described below. Antibodies can be added directly to the undiluted chromatin preparation for immunoprecipitation of chromatin complexes.

Before starting:

  • Remove and warm 200X Protease Inhibitor Cocktail (PIC). Make sure PIC is completely thawed.
  • Remove and warm 10X ChIP Buffer and ensure SDS is completely in solution.
  • Thaw digested chromatin preparation (from Step 9 in Section III) and place on ice.
  • Prepare low salt wash: 3 ml 1X ChIP Buffer (300 μl 10X ChIP Buffer + 2.7 ml water) per immunoprecipitation. Store at room temperature until use.
  • Prepare high salt wash: 1 ml 1X ChIP Buffer (100 μl 10X ChIP Buffer + 900 μl water) + 70 μl 5M NaCl per immunoprecipitation. Store at room temperature until use.
  1. In one tube, prepare enough 1X ChIP Buffer for the dilution of digested chromatin into the desired number of immunoprecipitations: 400 μl of 1X ChIP Buffer (40 μl of 10X ChIP Buffer + 360 μl water) + 2 μl 200X PIC per immunoprecipitation. When determining the number of immunoprecipitations, remember to include the positive control Histone H3 (D2B12) XP® Rabbit mAb #4620 and negative control Normal Rabbit IgG antibody samples. Place mix on ice.
  2. To the prepared 1X ChIP buffer, add the equivalent of 100 μl (5 to 10 μg of chromatin) of the digested, cross-linked chromatin preparation (from Step 9 in Section III) per immunoprecipitation. For example, for 10 immunoprecipitations, prepare a tube containing 4 ml 1X ChIP Buffer (400 μl 10X ChIP Buffer + 3.6 ml water) + 20 μl 200X PIC + 1 ml digested chromatin preparation.
  3. Remove a 10 μl sample of the diluted chromatin and transfer to a microfuge tube. This is your 2% Input Sample, which can be stored at -20°C until further use (Step 1 in Section VI).
  4. For each immunoprecipitation, transfer 500 μl of the diluted chromatin to a 1.5 ml microcentrifuge tube and add the immunoprecipitating antibody. The amount of antibody required per IP varies and should be determined by the user. For the positive control Histone H3 (D2B12) XP® Rabbit mAb #4620, add 10 μl to the IP sample. For the negative control Normal Rabbit IgG, add 1 μl (1 μg) to 2 μl (2 μg) to the IP sample. Incubate IP samples 4 h to overnight at 4°C with rotation.
  5. Resuspend ChIP-Grade Protein G Agarose Beads by gently vortexing. Immediately add 30 μl of Protein G Agarose Beads to each IP reaction and incubate for 2 h at 4°C with rotation.
  6. Pellet Protein G Agarose Beads in each immunoprecipitation by brief 1 min centrifugation at 6,000 rpm in a microcentrifuge and remove supernatant.
  7. Wash Protein G Agarose Beads by adding 1 ml of low salt wash to the beads and incubate at 4°C for 5 min with rotation. Repeat steps 6 and 7 two additional times for a total of 3 low salt washes.
  8. Add 1 ml of high salt wash to the beads and incubate at 4°C for 5 min with rotation.
  9. Pellet Protein G Agarose Beads in each immunoprecipitation by brief 1 min centrifugation at 6,000 rpm in a microcentrifuge. Remove supernatant and immediately proceed to Section VI.

VI. Elution of Chromatin from Antibody/Protein G Agarose Beads and Reversal of Cross-links:

Before starting:

  • Remove and warm 2X ChIP Elution Buffer in a 37°C water bath and ensure SDS is in solution.
  • Set a water bath or thermomixer to 65°C.
  • Prepare 150 μl 1X ChIP Elution Buffer (75 μl 2X ChIP Elution Buffer + 75 μl water) for each immunoprecipitation and the 2% input sample.
  1. Add 150 μl of the 1X ChIP Elution Buffer to the 2% input sample tube and set aside at room temperature until Step 6.
  2. Add 150 μl 1X ChIP Elution Buffer to each IP sample.
  3. Elute chromatin from the antibody/Protein G Agarose Beads for 30 min at 65°C with gentle vortexing (1,200 rpm). A thermomixer works best for this step. Alternatively, elutions can be performed at room temperature with rotation, but may not be as complete.
  4. Pellet Protein G Agarose Beads by brief 1 min centrifugation at 6,000 rpm in a microcentrifuge.
  5. Carefully transfer eluted chromatin supernatant to a new tube.
  6. To all tubes, including the 2% input sample from Step 1, reverse cross-links by adding 6 μl 5M NaCl and 2 μl Proteinase K, and incubate 2 h at 65°C. This incubation can be extended overnight.
  7. Immediately proceed to Section VII. Alternatively, samples can be stored at -20°C. However, to avoid formation of a precipitate, be sure to warm samples to room temperature before adding DNA Binding Reagent A (Section VII, Step 1).

VII. DNA Purification Using Spin Columns:

Before starting:

  • Add 12 ml of isopropanol to DNA Binding Reagent A and 24 ml of ethanol (96-100%) to DNA Wash Reagent B before use. These steps only have to be performed once prior to the first set of DNA purifications.
  • Remove one DNA purification spin column and collection tube for each DNA sample from Section VI.
  1. Add 600 μl DNA Binding Reagent A to each DNA sample and vortex briefly.
    • 4 volumes of DNA Binding Reagent A should be used for every 1 volume of sample.
  2. Transfer 375 μl of each sample from Step 1 to a DNA purification spin column in collection tube.
  3. Centrifuge at 14,000 rpm in a microcentrifuge for 30 sec.
  4. Remove the spin column from the collection tube and discard the liquid. Replace spin column in the collection tube.
  5. Transfer the remaining 375 μl of each sample from Step 1 to the spin column in collection tube. Repeat Steps 3 and 4.
  6. Add 700 μl of DNA Wash Reagent B to the spin column in collection tube.
  7. Centrifuge at 14,000 rpm in a microcentrifuge for 30 sec.
  8. Remove the spin column from the collection tube and discard the liquid. Replace spin column in the collection tube.
  9. Centrifuge at 14,000 rpm in a microcentrifuge for 30 sec.
  10. Discard collection tube and liquid. Retain spin column.
  11. Add 50 μl of DNA Elution Reagent C to each spin column and place into a clean 1.5 ml microcentrifuge tube.
  12. Centrifuge at 14,000 rpm in a microcentrifuge for 30 sec to elute DNA.
  13. Remove and discard DNA purification spin column. Eluate is now purified DNA. Samples can be stored at -20°C.

VIII. Quantification of DNA by PCR:

Recommendations:

  • Use Filter-tip pipette tips to minimize risk of contamination.
  • The control primers included in the kit are specific for the human or mouse RPL30 gene and can be used for either standard PCR or quantitative real-time PCR. If the user is performing ChIPs from another species, it is recommended that the user design the appropriate specific primers to DNA and determine the optimal PCR conditions.
  • A Hot-Start Taq polymerase is recommended to minimize the risk of non-specific PCR products.
  • PCR primer selection is critical. Primers should be designed with close adherence to the following criteria:
Primer length: 24 nucleotides
Optimum Tm: 60°C
Optimum GC: 50%
Amplicon Size: 150 to 200 bp (for standard PCR)
80 to 160 bp (for real-time quantitative PCR)

Standard PCR Method:

  1. Label the appropriate number of 0.2 ml PCR tubes for the number of samples to be analyzed. These should include the 2% input sample, the positive control histone H3 sample, the negative control normal rabbit IgG sample, and a tube with no DNA to control for DNA contamination.
  2. Add 2 μl of the appropriate DNA sample to each tube.
  3. Prepare a master reaction mix as described below, making sure to add enough reagent for two extra tubes to account for loss of volume. Add 18 μl of master mix to each reaction tube.
Reagent Volume for 1 PCR Reaction (18 μl)
Nuclease-free H2O 12.5 μl
10X PCR Buffer 2.0 μl
4 mM dNTP Mix 1.0 μl
5 µM RPL30 Primers 2.0 μl
Taq DNA Polymerase 0.5 μl
  1. Start the following PCR reaction program:
a. Initial Denaturation 95°C 5 min
b. Denature 95°C 30 sec
c. Anneal 62°C 30 sec
d. Extension 72°C 30 sec
e. Repeat Steps b-d for a total of 34 cycles.
f. Final Extension 72°C 5 min
  1. Remove 10 μl of each PCR product for analysis by 2% agarose gel or 10% poly-acrylamide gel electrophoresis with a 100 bp DNA marker. The expected size of the PCR product is 161 bp for human RPL30 and 159 bp for mouse RPL30.

Real-Time Quantitative PCR Method:

  1. Label the appropriate number of PCR tubes or PCR plates compatible with the model of PCR machine to be used. PCR reactions should include the positive control histone H3 sample, the negative control normal rabbit IgG sample, a tube with no DNA to control for contamination, and a serial dilution of the 2% input chromatin DNA (undiluted, 1:5, 1:25, 1:125) to create a standard curve and determine the efficiency of amplification.
  2. Add 2 μl of the appropriate DNA sample to each tube or well of the PCR plate.
  3. Prepare a master reaction mix as described below. Add enough reagents for two extra reactions to account for loss of volume. Add 18 μl of reaction mix to each PCR reaction tube or well.
Reagent Volume for 1 PCR Reaction (18 μl)
Nuclease-free H2O 6 μl
5 µM RPL30 Primers 2 μl
2X SYBR-Green Reaction Mix 10 μl
  1. Start the following PCR reaction program:
a. Initial Denaturation 95°C 3 min
b. Denature 95°C 15 sec
c. Anneal and Extension: 60°C 60 sec
d. Repeat steps b and c for a total of 40 cycles.
  1. Analyze quantitative PCR results using the software provided with the real-time PCR machine. Alternatively, one can calculate the IP efficiency manually using the Percent Input Method and the equation shown below. With this method, signals obtained from each immunoprecipitation are expressed as a percent of the total input chromatin.

Percent Input = 2% x 2(C[T] 2%Input Sample – C[T] IP Sample)

C[T] = CT = Threshold cycle of PCR reaction

APPENDIX A: Expected Chromatin Yield

When harvesting cross-linked chromatin from tissue samples, the yield of chromatin can vary significantly between tissue types. The table to the right provides a range for the expected yield of chromatin from 25 mg of tissue compared to 4 x 106 HeLa cells, and the expected DNA concentration, as determined in Section IV of the protocol. For each tissue type, disaggregation using a Medimachine (BD Biosciences) or a Dounce homogenizer yielded similar amounts of chromatin. However, chromatin processed from tissues disaggregated using the Medimachine typically gave higher IP efficiencies than chromatin processed from tissues disaggregated using a Dounce homogenizer. A Dounce homogenizer is strongly recommended for disaggregation of brain tissue, as the Medimachine does not adequately disaggregate brain tissue into a single-cell suspension. For optimal ChIP results, we recommend using 5 to 10 µg of digested, cross-linked chromatin per immunoprecipitation; therefore, some tissues may require harvesting more than 25 mg per each immunoprecipitation.

Tissue/Cell Total Chromatin Yield Expected DNA Concentration
Spleen 20-30 µg per 25 mg tissue 200-300 µg/ml
Liver 10-15 µg per 25 mg tissue 100-150 µg/ml
Kidney 8-10 µg per 25 mg tissue 80-100 µg/ml
Brain 2-5 µg per 25 mg tissue 20-50 µg/ml
Heart 2-5 µg per 25 mg tissue 20-50 µg/ml
HeLa 10-15 µg per 4 x 106 cells 100-150 µg/ml

APPENDIX B: Optimization of Chromatin Digestion

Optimal conditions for the digestion of cross-linked chromatin DNA to 150-900 base pairs in length is highly dependent on the ratio of Micrococcal Nuclease to the amount of tissue or number of cells used in the digest. Below is a protocol for determination of the optimal digestion conditions for a specific tissue or cell type.

  1. Prepare cross-linked nuclei from 125 mg of tissue or 2 X 107 cells (equivalent of 5 IP preps), as described in Sections I, II, and III. Stop after Step 2 of Section III and proceed as described below.
  2. Transfer 100 μl of the nuclei preparation into 5 individual 1.5 ml microcentrifuge tubes and place on ice.
  3. Add 3 μl Micrococcal Nuclease stock to 27 μl of 1X Buffer B + DTT (1:10 dilution of enzyme).
  4. To each of the 5 tubes in Step 2, add 0 μl, 2.5 μl, 5 μl, 7.5 μl, or 10 μl of the diluted Micrococcal Nuclease, mix by inverting tube several times and incubate for 20 min at 37°C with frequent mixing.
  5. Stop each digest by adding 10 μl of 0.5 M EDTA and placing tubes on ice.
  6. Pellet nuclei by centrifugation at 13,000 rpm in a microcentrifuge for 1 min at 4°C and remove supernatant.
  7. Resuspend nuclear pellet in 200 μl of 1X ChIP buffer + PIC. Incubate on ice for 10 min.
  8. Sonicate lysate with several pulses to break nuclear membrane. Incubate samples 30 sec on wet ice between pulses. Optimal conditions required for complete lysis of nuclei can be determined by observing nuclei under light microscope before and after sonication. HeLa nuclei were completely lysed after 3 sets of 20-sec pulses using a VirTis Virsonic 100 Ultrasonic Homogenizer/Sonicator set at setting 6 with a 1/8-inch probe. Alternatively, nuclei can be lysed by homogenizing the lysate 20 times in a Dounce homogenizer; however, lysis may not be as complete.
  9. Clarify lysates by centrifugation at 10,000 rpm in a microcentrifuge for 10 min at 4°C.
  10. Transfer 50 μl of each of the sonicated lysates to new microfuge tubes.
  11. To each 50 μl sample, add 100 μl nuclease-free water, 6 μl 5 M NaCl and 2 μl RNAse A. Vortex to mix and incubate samples at 37°C for 30 min.
  12. To each RNAse A-digested sample, add 2 μl Proteinase K. Vortex to mix and incubate sample at 65°C for 2 h.
  13. Remove 20 μl of each sample and determine DNA fragment size by electrophoresis on a 1% agarose gel with a 100 bp DNA marker.
  14. Observe which of the digestion conditions produces DNA in the desired range of 150-900 base pairs (1 to 5 nucleosomes, see Figure 3). The volume of diluted Micrococcal Nuclease that produces the desired size of DNA fragments using this optimization protocol is equivalent to 10 times the volume of Micrococcal Nuclease stock that should be added to one immunoprecipitation preparation (25 mg of disaggregated tissue cells or 4 X 106 tissue culture cells) to produce the desired size of DNA fragments. For example, if 5 μl of diluted Micrococcal Nuclease produces DNA fragments of 150-900 base pairs in this protocol, then 0.5 μl of stock Micrococcal Nuclease should be added to one immunoprecipitation preparation during the digestion of chromatin in Section III.
  15. If results indicate that DNA is not in the desired size range, then repeat optimization protocol, adjusting the amount of Micrococcal Nuclease in each digest accordingly. Alternatively, the digestion time can be changed to increase or decrease the extent of DNA fragmentation.

APPENDIX C: Troubleshooting Guide

Problem Possible Causes Recommendation
1. Concentration of the digested chromatin is too low (low chromatin yield). Not enough tissue or cells were added to the chromatin digestion or cell nuclei were not completely lysed after digestion. Add additional chromatin to each IP to give at least 5 μg/IP and continue with protocol.
Weigh tissue or count a separate plate of cells prior to cross-linking to determine accurate cell number. Some tissues may require processing of more than 25 mg per IP. The amount of tissue can be increased to 50 mg per IP, while still maintaining efficient chromatin fragmentation and extraction
Increase the number of sonications following chromatin digestion. Visualize cell nuclei under microscope before and after sonication to confirm complete lysis of nuclei.
2. Chromatin is under-digested and fragments are too large (greater than 900 bp). Large chromatin fragments can lead to increased background and lower resolution. Too many cells or not enough Micrococcal Nuclease was added to the chromatin digestion. Weigh tissue or count a separate plate of cells prior to cross-linking to determine accurate cell number. Add less tissue or cells, or more Micrococcal nuclease to the chromatin digest. See Appendix B for optimization of chromatin digestion.
Tissue or cells may have been over cross-linked. Cross-linking for longer than 10 min may inhibit digestion of chromatin. Perform a time course at a fixed formaldehyde concentration. Shorten the time of cross-linking to 10 min or less.
3. Chromatin is over-digested and fragments are too small (exclusively 150 bp mono-nucleosome length). Complete digestion of chromatin to mono-nucleosome length DNA may diminish signal during PCR quantification, especially for amplicons greater than 150 bp in length. Not enough cells or too much Micrococcal Nuclease added to the chromatin digestion. Weigh tissue or count a separate plate of cells prior to cross-linking to determine accurate cell number. Add more tissue or cells, or less Micrococcal nuclease to the chromatin digest. See Appendix B for optimization of chromatin digestion.
4. No product or very little product in the input PCR reactions. Not enough DNA added to the PCR reaction or conditions are not optimal. Add more DNA to the PCR reaction or increase the number of amplification cycles.
PCR amplified region may span nucleosome-free region. Optimize the PCR conditions for experimental primer set using purified DNA from cross-linked and digested chromatin. Design a different primer set and decrease length of amplicon to less than 150 bp (see primer design recommendations in Section VIII).
Not enough chromatin added to the IP or chromatin is over-digested. For optimal ChIP results, add 5 to 10 μg chromatin per IP. See recommendations for problems 1 and 3 above.
5. No product in the positive control histone H3-IP RPL30 PCR reaction. Not enough chromatin or antibody added to the IP reaction or IP incubation time is too short. Be sure to add 5 to 10 μg of chromatin and 10 μl of antibody to each IP reaction and incubate with antibody over-night and an additional 2 h after adding Protein G beads.
Incomplete elution of chromatin from Protein G beads. Elution of chromatin from Protein G beads is optimal at 65°C with frequent mixing to keep beads suspended in solution.
6. Quantity of product in the negative control Rabbit IgG-IP and positive control histone H3-IP PCR reactions is equivalent (high background signal). Too much or not enough chromatin added to the IP reaction. Or too much antibody added to the IP reaction. For optimal ChIP results, add 5 to 10 µg of chromatin and 10 μl of histone H3 antibody to each IP reaction. Reduce the amount of normal rabbit IgG to 1 μl per IP.
Too much DNA added to the PCR reaction or too many cycles of amplification. Add less DNA to the PCR reaction or decrease the number of PCR cycles. It is very important that the PCR products are analyzed within the linear amplification phase of PCR. Otherwise, the differences in quantities of starting DNA cannot be accurately measured. Alternatively, quantify immunoprecipitations using real-time quantitative PCR.
7. No product in the Experimental Antibody-IP PCR reaction. Not enough DNA added to the PCR reaction. Add more DNA to the PCR reaction or increase the number of amplification cycles.
Not enough antibody added to the IP reaction. Typically a range of 1 to 5 μg of antibody are added to the IP reaction; however, the exact amount depends greatly on the individual antibody. Increase the amount of antibody added to the IP.
Antibody does not work for ChIP. Find an alternate antibody source.

posted December 2011

revised November 2013

protocol id: 65

Product Includes Volume Storage Temp
Glycine Solution (10X) 100 ml 4°C
Buffer A (4X) 25 ml 4°C
Buffer B (4X) 25 ml 4°C
ChIP Buffer (10X) 20 ml 4°C
ChIP Elution Buffer (2X) 7 ml 4°C
5 M NaCl 3 ml 4°C
ChIP-Grade Protein G Agarose Beads 9007 1 ml 4°C
DNA Binding Reagent A (add 12 ml isopropanol before use) 12 ml RT
DNA Wash Reagent B (add 24 ml ethanol before use) 6 ml RT
DNA Elution Reagent C 1 ml RT
DNA Spin Columns 36 Pack RT
Protease Inhibitor Cocktail (200X) 750 µl -20°C
RNAse A (10 mg/ml) 50 µl -20°C
Micrococcal Nuclease 10011 60 µl -20°C
Proteinase K 100 µl -20°C
SimpleChIP® Human RPL30 Exon 3 Primers 7014 150 µl -20°C
SimpleChIP® Mouse RPL30 Intron 2 Primers 7015 150 µl -20°C
Histone H3 (D2B12) XP® Rabbit mAb (ChIP Formulated) 4620 100 µl -20°C
Normal Rabbit IgG 2729 50 µl -20°C
DTT (Dithiothreitol) 200 µl -20°C

Product Description

The SimpleChIP® Plus Enzymatic Chromatin IP Kit (Agarose Beads) contains the buffers and reagents necessary to perform up to 30 chromatin immunoprecipitations from cells or tissue samples, and is optimized for 4 X 106 cells or 25 mg of tissue per immunoprecipitation. A complete assay can be performed in as little as two days and can easily be scaled up or down for use with more or less cells or tissue sample. Cells or tissue are fixed with formaldehyde and lysed, and chromatin is fragmented by partial digestion with Micrococcal Nuclease to obtain chromatin fragments of 1 to 5 nucleosomes. Enzymatic fragmentation of chromatin is much milder than sonication and eliminates problems resulting from variability in sonication power and emulsification of chromatin during sonication, which can result in incomplete fragmentation of chromatin or loss of antibody epitopes due to protein denaturation and degradation. Chromatin immunoprecipitations are performed using ChIP-validated antibodies and ChIP-Grade Protein G Agarose Beads. After reversal of protein-DNA cross-links, the DNA is purified using DNA purification spin columns, allowing for easy and efficient recovery of DNA and removal of protein contaminants without the need for phenol/chloroform extractions and ethanol precipitations. The enrichment of particular DNA sequences during immunoprecipitation can be analyzed by standard PCR, quantitative real-time PCR, or amplification for ChIP on chip, sequencing or cloning techniques. The SimpleChIP® Plus Kit also provides important controls to ensure a successful ChIP experiment. The kit contains a positive control Histone H3 Antibody, a negative control Normal Rabbit IgG Antibody and primer sets for PCR detection of the human and mouse ribosomal protein L30 (RPL30) genes. Histone H3 is a core component of chromatin and is bound to most DNA sequences throughout the genome, including the RPL30 locus. Thus, the Histone H3 Antibody provides a universal positive control that should enrich for almost any locus examined.


Specificity / Sensitivity

The SimpleChIP® Plus Enzymatic Chromatin IP Kit can be utilized with any ChIP-validated antibody to detect endogenous levels of protein-DNA interactions and histone modifications in mammalian cells and tissue samples (see Figures 1 and 2). The positive control Histone H3 Antibody recognizes many different species of the highly conserved Histone H3 protein, including human, mouse, rat and monkey. Primer sets are included for the human and mouse positive control RPL30 gene loci; however, the use of other species with the kit requires the design of additional control primer sets.


The chromatin immunoprecipitation (ChIP) assay is a powerful and versatile technique used for probing protein-DNA interactions within the natural chromatin context of the cell (1,2). This assay can be used to identify multiple proteins associated with a specific region of the genome, or the opposite, to identify the many regions of the genome bound by a particular protein (3-6). It can be used to determine the specific order of recruitment of various proteins to a gene promoter or to "measure" the relative amount of a particular histone modification across an entire gene locus (3,4). In addition to histone proteins, the ChIP assay can be used to analyze binding of transcription factors and co-factors, DNA replication factors and DNA repair proteins. When performing the ChIP assay, cells or tissues are first fixed with formaldehyde, a reversible protein-DNA cross-linking agent that "preserves" the protein-DNA interactions occurring in the cell (1,2). Cells are lysed and chromatin is harvested and fragmented using either sonication or enzymatic digestion. The chromatin is then immunoprecipitated with antibodies specific to a particular protein or histone modification. Any DNA sequences that are associated with the protein or histone modification of interest will co-precipitate as part of the cross-linked chromatin complex and the relative amount of that DNA sequence will be enriched by the immunoselection process. After immunoprecipitation, the protein-DNA cross-links are reversed and the DNA is purified. Standard PCR or Quantitative Real-Time PCR can be used to measure the amount of enrichment of a particular DNA sequence by a protein-specific immunoprecipitation (1,2). Alternatively, the ChIP assay can be combined with genomic tiling micro-array (ChIP on chip) techniques, high throughput sequencing, or cloning strategies, all of which allow for genome-wide analysis of protein-DNA interactions and histone modifications (5-8).


1.  Orlando, V. (2000) Trends Biochem Sci 25, 99-104.

2.  Kuo, M.H. and Allis, C.D. (1999) Methods 19, 425-33.

3.  Agalioti, T. et al. (2000) Cell 103, 667-78.

4.  Soutoglou, E. and Talianidis, I. (2002) Science 295, 1901-4.

5.  Mikkelsen, T.S. et al. (2007) Nature 448, 553-60.

6.  Lee, T.I. et al. (2006) Cell 125, 301-13.

7.  Weinmann, A.S. and Farnham, P.J. (2002) Methods 26, 37-47.

8.  Wells, J. and Farnham, P.J. (2002) Methods 26, 48-56.



For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc.
SimpleChIP® is a trademark of Cell Signaling Technology, Inc.