Encyclopedia of Experiments: Cancer Research
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Transcript
- Chromatin immunoprecipitation, or ChIP, is a technique used to determine protein-DNA interactions in cells. Proteins bind to specific sites on DNA and regulate gene expression. First, stabilize the DNA-protein complex with formaldehyde. Formaldehyde cross-links proteins to the associated DNA sequence by forming covalent bonds.
Next, use a suitable lysis buffer to lyse the cells to release chromatin containing the cross-linked DNA-protein complexes. Centrifuge the suspension and sonicate the supernatant to obtain chromatin fragments. Subsequently, label the sample with a suitable antibody that binds specifically to the DNA-bound protein.
Add specialized magnetic beads to the sample. The bead recognizes the antibody and binds to it. Use a magnet to collect the magnetic beads with the antibody-protein-DNA complex. Wash the sample to remove unbound chromatin or antibodies. Add a high-salt concentration buffer to the complex and incubate at higher temperatures to reverse DNA protein cross-links.
Using a magnet, collect the supernatant containing the DNA and proteins. Add proteinase K enzyme to degrade the proteins in the sample. Add a suitable buffer to precipitate DNA and use it for further analysis. The following protocol will demonstrate the chromatin immunoprecipitation in chronic lymphocytic leukemia cells to identify NFAT2 target genes.
- First, prepare 20 microliters of protein A coated beads for each precipitation in one 1.5 milliliter tube. Allow the beads to settle on a magnetic rack for one minute and remove the supernatant. Then wash the beads with 40 microliters of 1x ChIP buffer 1 for each 20 microliters of protein A coated beads by pipetting up and down.
Allow the beads to settle on a magnetic rack for one minute and remove the supernatant. Repeat this washing process three more times before resuspending the beads in their original volume of 1x ChIP buffer 1. Add BSA, protease inhibitor, 5x ChIP buffer, and ChIP-seq grade water to the beads. Next, add the sheared chromatin.
Use 10 micrograms of anti-NFAT2 antibody to capture NFAT2 and 2.5 micrograms of IgG antibody as a control. Incubate the mixtures overnight at 4 degrees Celsius on a wheel rotating at six RPM. The next day, spin the tubes for five seconds at 7,000 times g and incubate them on the magnetic rack for one minute.
Then, aspirate the supernatant and wash the beads once with wash buffers 1, 2, 3, and 4, consecutively. Spin the tubes for five seconds at 7,000 times g before incubating the tubes on the magnetic rack for one minute. After this, remove the supernatant and add the next wash buffer.
After the last wash, spin the tubes for five seconds at 7,000 times g. Incubate the tubes on the magnetic rack for one minute. Remove the supernatant and take up the beads in 100 microliters of elution buffer 1. Then, incubate the tubes on the rotating wheel for 30 minutes.
Next, briefly spin the tubes and place them on a magnetic rack for one minute. Transfer the supernatant to a new 1.5 milliliter tube and add 4 microliters of elution buffer 2. To create the input control, mix 1 microliter of the sheared chromatin with 99 microliters of elution buffer one and 4 microliters of elution buffer two. Then incubate the samples for four hours at 65 degrees Celsius with shaking.
After this, add 100 microliters of 100% isopropanol to the tubes. Vortex and spin the samples for five seconds at 7,000 times g. Then add 10 microliters of magnetic beads to each sample. Vortex and incubate the samples on a rotating wheel at room temperature for an hour.
Next, spin the tubes briefly and place them on a magnetic rack for one minute. Aspirate the supernatant and resuspend the beads in 100 microliters of wash buffer one. Invert the tubes to mix and incubate them for five minutes on a rotating wheel at room temperature. Next briefly centrifuge the tubes. Place them in a magnetic rack for one minute and use a pipette to remove the supernatant.
Then, add 100 microliters of wash buffer two. Invert the tubes to mix, and incubate them for five minutes on a rotating wheel at room temperature. After this, briefly centrifuge the tubes and place them in a magnetic rack for one minute. Remove the wash buffer two, via aspiration, and resuspend the beads in 55 microliters of elution buffer.
To elute the precipitated DNA, incubate the samples in a rotating wheel at room temperature for 15 minutes. After this, spin the tubes briefly and place them in a magnetic rack for one minute. Finally, transfer the supernatant to new 1.5 milliliter tubes.
