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Native ChIP Protocol

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Native ChIP Protocol

Native ChIP is a technique used for studying naturally occurring protein-DNA interactions. It is usually specific to histone modifications and native chromatin is used as the starting chromatin. Micrococcal nuclease first cuts the chromatin linker DNA, which yields fragments of intact nucleosomes from 200bp-1000bp. The DNA fractions of interest can be pulled down by the specific antibody-antigen reactions. Therefore, this process enriches for the DNA fragments bound by target proteins. Finally, DNA can be purified from the complex and analyzed with PCR or qPCR protocols.


Sandwich ELISA Diagram

Native ChIP diagram courtesy of He C. and Bonasio R. Click to enlarge.



Solution and reagents

Reaction buffer:

1mM CaCl2, 0.2% Triton X-100 or NP-40, 50mM Tris-HCl (pH 7.6)

RIPA buffer:

0.1% SDS, 0.1%NaDOC, 1% Triton X-100, 1mM EDTA, 10mM Tris-HCl (pH 7.6)

LiCl buffer:

0.25M LiCl, 0.5% NP-40, 0.5% NaDOC, 10mM Tris-HCl (pH 7.6)

TE buffer:

1mM EDTA, 10mM Tris-HCl (pH 7.6)

 

Sample preparation

Use cell culture from 10cm culture dishes with 20mL of medium. Cells are ready to be used for a ChIP assay when the density of cells reaches 80%-90%. The abundance of target protein depends upon the protein of interest and the number of cells. Refer to the table below:

Protein

Number of cells (for a ChIP reaction)

Protein target abundance

Histone protein
RNA polymerase II

104

high

Transcription factor

105-106

medium

Cofactor

107 or more

low

 

For optimal results, a starting concentration of >4×106 cells for histone protein and RNA polymerase II is recommended. Transcription factors or cofactors may require more cells.

  1. Take out the culture dishes and discard the culture medium.
  2. Wash the cells with ice-cold PBS three times.
  3. Add 1ml of PBS and collect the cells by scraping the cells from culture dishes.
  4. Centrifuge for 3min, 4 ℃ at 2500 RPM to collect the cells.
  5. Resuspend the cells by adding 500μl of reaction buffer and fresh protease inhibitors to lyse the cells on ice for 10 min.
  6. Centrifuge for 3min, 4 ℃ at 2500 RPM to collect the cells.
  7. Resuspend the pellet by adding 1ml reaction buffer and micrococcal nuclease to react at 37 ℃ for 20 min. Refer to the instructions of the micrococcal nuclease to determine the enzymolysis condition. Determine the optimal processing time and concentration of micrococcal nuclease with a previous preliminary experiment.
  8. Add 5mM EDTA to terminate the enzymatic hydrolysis

Sonication

Sonication conditions may differ depending on the sonicator and type of cells used. Depending on the different cell lines, the ideal fragment size of DNA after sonication is 200-1000bp. See our Sonication Application protocol to determine the ideal size for your experiment.

  1. Sonicate the cells to obtain the ideal fractions of DNA is 200-1000bp. Sonication conditions may differ depending on the sonicator or type of cells used.
  2. Centrifuge for 2min, 4℃ at 2500 RPM. Transfer the supernatant into a new tube.
  3. To verify sonication results, 5μl of sheared chromatin supernatant can be used for agarose gel analysis/Bioanalyzer.

Immunoprecipitation

  1. Transfer 500μl of cell lysate into a new tube.
  2. Prepare four groups of sample conditions :
    Experimental group: add 2-5μg of specific antibody into 500μl cell lysate and place tube on a rotary mixer at 4℃ overnight to form the antigen-antibody complex.
    Input group: follow the same procedure as the experimental group. However, do not add antibodies.
    Negative control: follow the same procedure as the experimental group, but add a rabbit IgG antibody control.
    Positive control: follow the same procedure as the experimental group but add a histone H3 or RNA polymerase antibody control.
  3. The next day, add 50μl of magnetic beads to the complex solution and incubate at 4 oC for 2 to 4 hours.
  4. Place tube on magnetic stand and remove the supernatant.

Wash the beads

Wash the beads with filtered tips:

a. 2×1ml RIPA buffer
b. 2×1ml RIPA buffer +0.3M NaCl
c. 2×1ml LiCl buffer
d. 2× 1ml TE buffer + 0.2%Triton X-100
e. 1× 1ml TE buffer. Mix 1 min on rotator.

Wash the beads two times with the buffers above. Pipette 3-8 times by tips and rotate for 10 min each.

  1. Remove the wash buffer by placing tubes on a magnetic stand.
  2. Re-suspend the precipitation complex in 100μl TE buffer. Add 3-5ul of 10% SDS and 5ul of 20mg/ml proteinase K. Incubate at 65 ℃ overnight.
  3. The next day, vortex briefly. Place sample tubes on magnetic stand and transfer supernatant to a new tube .
  4. Wash beads with 100μl TE buffer + 0.5M NaCl. Combine with the surpernatant in step 4.

DNA purification

DNA can be extracted by using the EpiNext DNA Purification HT System and following the procedure found in the user guide.

Detection

Perform a PCR or Real time PCR assay to analyze the DNA associated with the target protein.


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