Application Verification Testing for Chromatin Immunoprecipitation

Thermo Fisher Scientific is committed to antibody performance and specificity testing. To support this commitment, each Invitrogen antibody that is indicated for chromatin immunoprecipitation (ChIP) applications has been tested using a protocol similar to that provided below. These tests help confirm antibody performance and help ensure superior results when used in experiments.

The typical ChIP protocol that Invitrogen antibodies are subjected to is reproduced below. This protocol includes:

• Preparation of required solutions
• Cell harvesting and cross linking
• Sonication
• Immunoprecipitation

Note: Some of the steps in this protocol require optimization depending on the sample and antibody being used.

1. For adherent cells, aspirate the media and wash cells with 10 mL of 1X PBS at room temperature.  
2. Aspirate the PBS and add enough trypsinizing reagent to cover the cells.
3. Incubate at 37°C for 2-5 min or until cells start to dislodge from the plate surface. 
4. When the cells have partially detached quench the trypsin with media and spin at 500 x g for 5 min. Remove the supernatant.
5. Add 10 mL of 1X PBS at room-temperature and gently pipette the cells up and down to mix.
   
   Note:For suspension cells directly proceed from step 4.
   
   
6. Transfer the cell suspension to a 15 mL conical tube and spin at 500 × g for 5 min to pellet cells. 
7. Discard supernatant and re-suspend the pellet in 1X PBS at room-temperature. 
8. Collect a small aliquot to verify that the cells are at the desired density. 
9. Determine the volume of cell suspension required (10-20 million cells per 5 mL of 1X PBS) 
10. Add formaldehyde to a final concentration of 1% (v/v) to the cell suspension. For example, add 135 µL of 37% formaldehyde to 5 mL of cell suspension.
11. Invert the tube to mix, and rotate gently for 10-15 min at room temperature
    
    Note: Perform a time course analysis to determine optimal incubation time for your experiment. 
    
    
12. Quench the formaldehyde by adding 550 µL of 1.25 M Glycine to a final concentration of 125 mM. Gently rotate the cells for 5 min at room temperature.
13. Centrifuge the crosslinked cells at 1000 × g for 5 min at 4°C.  
14. Discard supernatant and re-suspend the cells in 5 mL of ice-cold 1X PBS, and spin at 1000 × g for 5 min at 4°C. 
15. Discard supernatant and repeat wash with ice-cold 1X PBS. 
16. Re-suspend the cell pellet in cell lysis buffer (1 mL per 2 x107 cells) containing protease & phosphatase inhibitor cocktail. Incubate for 10 min on ice and centrifuge at 2000 × g for 10 min at 4°C.
17. Discard supernatant, and re-suspend the pellet in nuclear lysis buffer containing protease & phosphatase inhibitor cocktail (500 µL per 2 x107 cells) and the place the sample on ice.

1. Shear the chromatin using a bath or probe sonicator to generate ~200–500 bp fragments for qPCR and ~100–300 bp fragments for massive parallel DNA sequencing analysis. In general, sonication conditions must be optimized for each sample type and instrument.
   
   Optional: To evaluate the size of the DNA fragments, add 170 µL of dilution buffer and 10 µL of 5 M NaCl to 20 µL of sheared chromatin, and incubate at 65°C overnight. The next day, add 1 µL of RNase A (10 mg/mL) and incubate for 30 min at 37°C. Following RNase A treatment, add 5 µL of Proteinase K (20 mg/mL) and heat at 55°C for 0.5-1 h. Purify DNA using either a DNA purification column or phenol-chloroform extraction and proceed to agarose gel electrophoresis.
   
   
2. Centrifuge at maximum speed (15000 - 20000 × g) for 10 min and collect the supernatant in a fresh tube. This is your sheared chromatin.
3. Proceed to immunoprecipitation or snap-freeze the chromatin in liquid nitrogen or on dry ice and store frozen aliquots at –80°C.

1. Take 25-100 µL (equivalent to 1-2 million cells) of sheared chromatin and dilute with 9-fold volume of ice-cold dilution buffer containing protease & phosphatase inhibitor cocktails. Store 1/10th volume of the chromatin used per IP at -20°C. This chromatin (INPUT) will be used as a reference for ChIP qPCR. 
2. Add primary antibody (2-5 µg) to the diluted chromatin and rotate end over end overnight at 4°C. In parallel, use an equal amount of Rabbit or Mouse IgG antibodies as negative IP controls to measure non-specific binding.
3. The next day, add 20 -30 µL of Dynabeads (Protein A/G beads) to each of the samples and incubate for 1-2 hr on rotation at 4°C.
4. Wash beads sequentially with 500 µL of low salt buffer, high salt buffer, LiCl buffer and TE buffer by rotating end over end at room temperature each for 5 min. Then, separate the beads using DynaMag-PCR Magnet.
5. After the final wash, remove the supernatant and re-suspend the beads with 100 µL of elution buffer and rotate end over end at room temperature for 20 min.
6. Spin the tubes briefly, and then place them in the DynaMag-PCR Magnet and transfer the supernatant to a fresh tube. Do not discard supernatant—this contains your sample. 
7. At this stage, take out the stored INPUT and make up the volume to 100 µL with elution buffer.
8. Add 5 µL of 5 M NaCl to both IP and INPUT samples, and incubate at 65°C for 3-4 hr.
9. Add 1 µL of RNase A (10 mg/mL) to each sample, and incubate at 37°C for 30 min.
10. Add 5 µL of Proteinase K (20 mg/mL) to each sample, and incubate at 55°C for 30 – 60 min.
11. Purify DNA using either DNA purification columns or phenol-chloroform extraction.
12. Store the purified DNA at -20°C or proceed with ChIP qPCR.