The following method offers reliable and sensitive detection of specific chromatin modifications on selected plant genes. First cross-link modified histones and DNA with formaldehyde. Then extract and sonicate the chromatin to facilitate immunoprecipitation with modification specific antibodies.
Finally analyze the chip reaction by the dross linking of histone DNA complexes and gene specific real-time quantitative PCR. In plants, this approach can provide molecular insights like the specific histone modifications associated with C four photosynthesis in maze and systemic immunity in Arabidopsis. The main advantage of chromatin immunoprecipitation over existing methods like Mars spectrometry is that this techniques allows the detection of histon modifications on selected sequences into plant genome.
Generally, individuals new to this method will struggle because the protocol entails a couple of steps whose correct performance is key to success of the procedure. In this video, we use chip analysis to provide insight into deregulation of gene expression in the model plant Arabidopsis ANA demonstrating the procedure will be mic yakovich, a PhD student For each sample begin with a harvest of two grams of arabidopsis leaves and a 50 milliliter plastic tube. Fill the 40 milliliter mark with crosslinking buffer and position a tailored filter sponge right above the buffer surface to ensure the leaves stay immersed.
Now, place the samples in a desiccate with vacuum and filtrate for 10 minutes. To stop the crosslinking reaction, add 2.5 milliliters of two molar glycine and mixed by inversion. Then infiltrate again for five minutes, continue to vacuum, infiltrate for another five minutes.
Then discard the fluid while harvesting leaves on a sieve. After washing the leaves twice with one liter of water in a plastic beaker, dry the leaves thoroughly with paper towels collect leaves into a fresh plastic tube and freeze in liquid nitrogen. Store the samples at minus 80 degrees Celsius until chromatin isolation.
Using a liquid nitrogen cooled mortar and pestle, grind the frozen samples to a fine powder. Transfer the powder to a 50 milliliter plastic tube. Suspend the powder in 30 milliliters of extraction buffer number one, and incubate for 15 minutes at four degrees celsius on an overhead shaker.
Pass the suspension through four layers of mirror cloth into a fresh 50 milliliter plastic tube. After centrifugation, discard the SUP natin. Next, use a paintbrush to resuspend the pellet in 50 microliters of extraction buffer number two, then add another 950 microliters of extraction buffer.
Number two, transfer the suspension to a 1.5 milliliter micro fuge tube and centrifuge for 10 minutes. Meanwhile, prepare two milliliter micro fuge tubes containing 1.5 milliliters of extraction buffer. Number three.
Next, discard the supernatants to gradually suspend the pellet in 300 microliters of extraction buffer. Number three. First, add a small volume of buffer.
Number three, suspend the pellet with a paintbrush and then add the remaining buffer. Now carefully layer the samples on top of the prepared tubes of 1.5 milliliters of extraction buffer. Number three, ensuring the two phases won't mix centrifuge samples for one hour at 16, 000 times G at four degrees Celsius.
Discard the sup natin and suspend the chromatin pellet with a paint brush in 300 microliters of sonication buffer sonicate chromatin to a DNA size of approximately 400 base pairs. When sonicate, make sure chromatin will not be heated above approximately 30 degrees Celsius. To protect heat sensitive cross links, centrifuge the sonicate sample to precipitate unsolved material and harvest the supernatant to two milliliter micro fuge tubes containing protein agarose and antibody binding buffer.
Add 200 microliters of the sample chromatin preparation. Incubate the tubes for one hour on an overhead shaker. After centrifugation, harvest the sample supernatants and aliquot 30 microliters of protein.
A aros to fresh 1.5 milliliter tubes for determination of chromatin concentration of input material. Reserve 40 microliters of the sonicated chromatin. Then add 400 microliters of the sonicated chromatin suspension to the 30 microliters of protein aros and an appropriate amount of modification specific antibody incubate overnight on an overhead shaker at four degrees Celsius.
Harvest beads by ification for two minutes at 440 times G.Remove the supernatant and perform successive 10 minute washes of bead pellets with 900 microliters of the respective buffer on an overhead shaker. After the final wash, completely remove any remaining buffer. To free the DNA from the histones, add 400 microliters of D cross linking buffer to the beads and the 40 microliter input sample preserved earlier vortex for five minutes centrifuge samples, and incubate at 65 degrees Celsius overnight.
Now, purify the DNA with a commercial DNA purification kit and perform conventional locus specific real-time quantitative R-T-P-C-R. In this example expression of the Arabidopsis ANA PR two defense gene was induced by Benzo Diaz ole a synthetic analog of the plant hormone salicylic acid. Results demonstrate that activation of PR two expression is associated with an increase in the acetylation of lysine residues on histones H three and H four on the PR two promoter.
While attempting to this procedure, it's important to remember that addition of extraction buffer one to the leaf powder causes over pressure in the closed fcon tube. Please remember to open the tube once in a while. Also, remember not to remove paintbrush after suspension of pellets before addition of the next buffer.
After watching this video, you should have a good understanding of how to determine hisone modifications and plain levers where form aldehyde based cross-linking of DNA and histones, isolation of chromatin immunoprecipitation and locus specific DNA quantitation to answer key questions on the role of chromatin and its modifications in different fields of plant biology.
