Chromatin Immunoprecipitation (ChIP) Assay

Protein-DNA interaction plays a key role for cellular functions such as signal transduction, gene transcription, chromosome segregation, DNA replication and recombination, and epigenetic silencing. Identifying the genetic targets of DNA-binding proteins and knowing the mechanisms of protein-DNA interaction is important for understanding cellular processes.

Chromatin immunoprecipitation (ChIP) provides a useful tool for studying protein-DNA interactions. It allows for the detection of specific proteins bound to specific gene sequences in living cells using PCR (ChIP-PCR), microarrays (ChIP-chip), or sequencing (ChIP-seq). For example, measurement of the amount of methylated histone H3 at lysine 9 (meH3-K9) associated with a specific gene promoter region under various conditions can be achieved through a ChIP-PCR assay, while the recruitment of meH3- K9 to the promoters on a genome-wide scale can be detected by ChIP-chip or ChIP-sequencing.

An ideal ChIP method requires maximum sensitivity and minimal background levels. This method should also be able to enrich highly abundant protein/DNA complexes in a high-throughput format with a small amount of cells. Here we describe an optimized protocol that reduces non-specific background ChIP levels, allowing capture of low abundance protein/transcription factors and increasing the specific enrichment of target protein/DNA complexes.

Figure 1. The flow chart of chromatin immunoprecipitation (ChIP) assay.

Materials and Equipment

Reagent Preparation

• Working lysis buffer
  Add 6 µL of Protease Inhibitor Cocktail to every 10 mL of Lysis Buffer required.
• Working ChIP buffer
  Add 1 µL of Protease Inhibitor Cocktail to every 10 mL of ChIP Buffer required.

Sample Preparation

For adherent or monolayer cells

1. Culture cells to 80%-90% confluence on a 6-well plate or 100 mm dish, then trypsinize and collect them into a 15 mL conical tube.
2. Centrifuge the cells at 1000 rpm for 5 min. Discard the supernatant and count the cells in a hemocytometer.
3. Wash cells with 10 mL of PBS once by centrifuging at 1000 rpm for 5 min. Discard the supernatant.
   Note: For cells that are not cross-linked, go directly to Step 9.
4. Add 9 mL fresh cell culture medium containing formaldehyde to a final concentration of 1% to cells.
5. Incubate at room temperature (20-25°C) for 10 min on a rocking platform (50-100 rpm).
6. Add 1 mL of 1.25 M glycine for every 9 mL of cross-link solution.
7. Mix and centrifuge at 1000 rpm for 5 min.
8. Remove medium and wash cells once with 10 mL of ice-cold PBS by centrifuging at 1000 rpm for 5 min.
9. Add Working Lysis Buffer to resuspend the cell pellet (200 µL/1 x 10⁶ cells) and incubate on ice for 10 min.
10. Vortex vigorously for 10 sec then centrifuge at 3000 rpm for 5 min. Go to the “Chromatin Extraction” section.

For suspension cells

1. Collect cells into a 15 mL conical tube (2 x 10⁵ to 5 x 10⁵ cells are required for each ChIP reaction).
2. Centrifuge the cells at 1000 rpm for 5 min. Discard the supernatant and count cells in a hemocytometer.
3. Wash cells with 10 mL of PBS once by centrifugation at 1000 rpm for 5 min. Discard the supernatant.
   Note: For cells that are not cross-linked, go directly to Step 9.
4. Add 9 mL fresh cell culture medium containing formaldehyde to a final concentration of 1% to cells.
5. Incubate at room temperature (20-25°C) for 10 min on a rocking platform (50-100 rpm).
6. Add 1 mL of 1.25 M glycine for every 9 mL of cross-link solution.
7. Mix and centrifuge at 1000 rpm for 5 min.
8. Remove medium and wash cells once with 10 mL of ice-cold PBS by centrifuging at 1000 rpm for 5 min.
9. Add Working Lysis Buffer to resuspend the cell pellet (200 µL/1 x 10⁶ cells) and incubate on ice for 10 min.
10. Vortex vigorously for 10 sec and centrifuge at 3000 rpm for 5 min. Then go to the “Chromatin Extraction” section.

ChIP Reaction Procedure

Antibody binding to the strip wells

1. Predetermine the number of the strip wells required for your experiment. Carefully remove unneeded strip wells and place them back in the bag (seal the bag tightly and store at 4°C).
2. Set up the antibody binding reactions by adding the reagents to each well according to the following table.
   
   

   Reagents	Sample (µL)	Positive control (µL)	Negative control (µL)
   Antibody Buffer	50-80	50-80	50-80
   Antibodies of interest	0.5-2	0	0
   Anti-RNA Polymerase II	0	0.8	0
   Non-Immune IgG	0	0	0.8

   
   Note: The final amount of each component should be (a) antibodies of interest: 0.8 µg/well; (b) RNA polymerase II: 0.8 µg/well; and (c) non-immune IgG: 0.8 µg/well.
3. Seal the wells with Adhesive Covering Film Strips and incubate the wells at room temperature for 60-90 min on an orbital shaker (100 rpm). Cell

Chromatin extraction

1. Add ChIP Buffer to resuspend the chromatin pellet (100 μL/1 x 10⁶ cells).
2. Transfer the chromatin lysate to a 1.5 mL vial and incubate on ice for 10 min and vortex occasionally.

Chromatin shearing

1. Resuspend the chromatin lysate and shear by using one of the following methods.
   Waterbath Sonication: Use 50 μL of chromatin lysate per 0.2 mL tube or per PCR plate well. Shear 20 cycles under cooling condition, 15 sec On, 30 sec Off, each at 170-190 watts.
   Probe Sonication: Use 300 μL of chromatin lysate per 1.5 mL microcentrifuge tube. Perform sonication with a microtip, set to 25% power output. Sonicate 3-4 pulses of 10-15 sec each, followed by 30-40 sec rest on ice between each pulse.
   Note: 1) The chromatin lysate can also be sheared by various enzyme. 2) The conditions of cross-linked DNA shearing can be optimized based on cells, sonicator equipment, and enzyme concentration.
2. Centrifuge at 12,000 rpm at 4°C for 10 min after shearing.
3. Transfer supernatant to a new vial and the chromatin solution can now be used immediately or stored at -80°C.
   Note: The size of sonicated chromatin should be verified before starting the immunoprecipitation step. The length of sheared DNA should be between 100-700 bp.

ChIP reaction

1. Carefully peel away the Adhesive Covering Film on the antibody binding wells to avoid contamination between each well.
2. Remove the antibody reaction solution and Non-Immune IgG solution from each well and wash the wells one time with 150 µL of ChIP Buffer.
3. Set up the ChIP reactions by adding the reagents to the wells that are bound with antibodies (sample and positive control wells) or IgG (negative control well) according to the following table:
   
   

   Reagents	Sample (µL)	Positive control (µL)	Negative control (µL)
   ChIP Buffer	48-78	48-78	48-78
   Chromatin	10-40	10-40	10-40
   Enrichment Enhancer	2	2	2
   Blocker Solution	10	10	10
   Total	100	100	100

   
   Note: The final amount of chromatin should be 2 μg/well; Sonicated chromatin can be further diluted with ChIP Buffer to desired concentration. For histone samples with sufficient chromatin (> 0.5 μg), the Enrichment Enhancer is not required and 50-80 μL of ChIP Buffer can be used. For low abundance targets, 2 μL of Enrichment Enhancer and 88 μL of chromatin can be used without adding ChIP Buffer.
4. Cap wells with Strip Caps and incubate at room temperature for 60-90 min on an orbital shaker (100 rpm). For low abundance targets, the incubation time should extend to 2-3 h or at 4°C overnight.
5. Carefully remove the solution and wash each well with 200 μL of the Wash Buffer each time for a total of 4 washes.
6. Wash each well with 200 μL of the DNA Release Buffer one time by pipetting DNA Release Buffer in and out of the well.
7. Prepare RNase A solution by adding 1 μL of RNase A to 40 μL of DNA Release Buffer.
8. Add 40 μL of DNA Release Buffer + RNase A to each well, and then cover with Strip Caps.
9. Incubate the wells at 42°C for 30 min.
10. Add 2 μL of Proteinase K to each of the wells and re-cap the wells.
11. Incubate the wells at 60°C for 45 min.
12. Quickly transfer the DNA solution from each well to 0.2 mL strip PCR tubes. Cap the PCR tubes.
13. Incubate the PCR tubes containing DNA solution at 95°C for 15 min in a thermocycler.
14. Place the PCR tubes at room temperature. DNA prepared at this step can be directly used for PCR or go to Step 15 for further purification for use in ChIP-Seq.
15. Place a spin column into a 2 mL collection tube. Add 200 μL of DNA Binding Solution to the samples and transfer the mixed solution to the column. Centrifuge at 12,000 rpm for 30 sec.
16. Add 200 μL of 90% ethanol to the column, centrifuge at 12,000 rpm for 30 sec. Remove the column from the collection tube and discard the flowthrough.
17. Replace column to the collection tube. Add 200 μL of 90% ethanol to the column and centrifuge at 12,000 rpm for 30 sec.
18. Remove the column and discard the flowthrough. Replace column to the collection tube and wash the column again with 200 μL of 90% Ethanol at 12,000 rpm for 1 min.
19. Place the column in a new 1.5 mL vial. Add 20 μL of DNA Elution Buffer directly to the filter in the column and centrifuge at 12,000 rpm for 30 sec to elute the purified DNA.
20. Purified DNA is now ready for PCR, ChIP-chip and ChIP-seq use or storage at -20°C.
    Note: 1 For real time PCR analysis, we recommend the use of 1-2 μL of eluted DNA in a 20 μLPCR reaction. 2 In general, the amplification difference between “normal IgG control” and “positive control” may vary from 3 to 8 cycles, depending on experimental conditions.

Analysis

Real time PCR

1. Primer Design: The primers designed should meet the criteria for real time PCR. For example, the covered sequence region should be 50-150 bp in length. G/C stretches at 3’ ends of primers should be avoided.
2. PCR Reaction: Real time PCR can be performed using your own proven method.

Endpoint PCR

1. Primer Design: The primers designed should meet the criteria for endpoint PCR. For example, the covered sequence region should be 100-400 bp in length.
2. PCR Reaction: Endpoint PCR can be performed using your own proven method.
   Note: It is important to stop the PCR reaction at the exponential phase by setting up an appropriate number of PCR cycles in order to make a reliable comparison of enrichment efficiency obtained from different ChIP reactions. Thus, the optimized number of PCR cycles should be determined empirically.
3. PCR Product Analysis: Endpoint PCR products can be analyzed by separating amplicons on a 1-2% agarose gel, followed by staining with ethidium bromide and visulizing with UV-illumination.

For research use only. Not for any other purpose.