Hi, I'm Amish Matan and I'm from the laboratory of Dr.Sha Mc Barian here in the psychiatry department of UMass Medical School. Today I will show you a procedure for chromatin immunoprecipitation in postmortem human brain tissue. Though the following procedure is not very complicated.
What is most exciting is that until recently expression studies in the human brain were limited to simple quantitation of mRNA and protein. This particular technique will give us a better understanding of the mechanistic basis of gene expression in both normal and diseased human brain. So let's get started To immuno precipitate chromatin.
We started with previously dissected on frozen postmortem human brain tissue. It's important to use safety glasses and work in a designated area for dealing with human tissue. When processing these samples, the brain tissue is downed with five times the brain volume of down sink buffer.
Once all the tissue seems to have become homogenous within the solution, we can transfer it to a two mil tube After the tissue has been homogenized. Add five units per mil of micro cocal nuclease to the sample and mix the solution by pipetting up and down. Then place it on ice.
It is important to do the step quickly. Since micro cocal nuclease has the ability to act at even four degrees Celsius. Once all samples have been treated with micro cocal nuclease, we quickly move to the 37 degree Celsius water bath and incubate the samples for seven minutes.
After the seven minute incubation, we add 0.5 molar EDTA to a concentration of 10 millimoles to stop the micro cocal nuclease activity. So now we're ready to go onto the ization step. After we have micro cocal nuclease treated, our samples which contain our nucleosomal DNA will begin ization.
Nava will take a 15 mil Falcon tube into which we'll add 4.5 mils of 0.2 millimolar EDTA 2.5 microliters of 0.2 molar benadine, and 4.5 micros of 0.1 molar PMSF into this tube. We'll transfer our sample. Now we incubate our sample for one hour on ice while vortexing it every 10 minutes.
At the end of the hour long incubation, we will add 2.5 microliters of three molar DTT. The sample is vortex once more, then it is centrifuge in a swing bucket rotor at 3, 175 RCF for 10 minutes at four degrees Celsius. After centrifugation, the supernatant is distributed, so that 500 microliters are used as our input control, which would only contain genomic DNA, and the rest is split into two tubes containing 1, 600 microliters of sample each, which will be our chromatin immunoprecipitation samples.
The input control is placed at minus 80 degrees Celsius overnight until further use. Next, we add 160 microliters of 10 XFSB one 10th of volume of our sample. Along with it, we add four micrograms of the antibody.
Now vortex the samples, and then rotate them at four degrees Celsius overnight. After the overnight incubation, we will wash the protein giros that will be used to isolate our nucleosomal DNA. Since these agro beads are very sensitive, it is necessary to cut off the heads of the pipette tips when working with them.
This is to ensure that the beads have more room while pipetting. Now add 1.6 mils of one XFS bead to 245 microliters of protein jaguars into a two mil micro centrifuge tube. This should be enough for four tubes of sample.
Next, separate the bead solution into two two mil tubes and refill each up to 1.6 mils with one XFSB. Rotate the tubes at room temperature for 30 seconds on a rotator and then centrifuge them at 0.1 RCF for 30 seconds. After centrifugation, remove the ATE using a vacuum.
Add 1.6 mils of one XFSB. Once again, rotate the samples for 30 seconds on a rotator and then centrifuge them again at 0.1 RCF FS for 30 seconds. After centrifugation, we remove the supernatant once again and combine both tubes with 1.5 mils of one XFSB.
Also add 15 microliters sperm DNA. Now rotate at room temperature for 30 minutes, and then centrifuge at 0.1 RCF for 30 seconds. Remove the supernatant and add 200 microliters of one XFSB.
Now add 90 microliters of agro speeds into each chromatin precipitation sample, and then rotate them at four degrees for one hour. For a negative control, add one mils of one XFSB into the remaining acro speeds and rotate it along with the chromatin immunoprecipitation sample at four degrees Celsius for one hour. During the one hour long incubation, we can take the opportunity to make some fresh resolution buffer.
This will be used later on in the experiment after the hour long. Incubation centrif feature the samples and negative control at 0.1 CFS for 30 seconds and discard the supernatant. Next, we will wash the beads with four different types of washing solutions, a low salt washing buffer, a high salt washing buffer, a lithium chloride solution, and finally, TE buffer at pH eight.
For each buffer except the lithium chloride solution, we will rotate the samples for three minutes at room temperature and then centrifuge them at 0.1 R Cs for 30 seconds. For the lithium chloride wash, rotate the samples at room temperature for only one minute. After each wash, discard the supinate and using a vacuum.
Now we're ready to go on to elute our samples. To begin the elution step, we add 250 microliters of freshly prepared elu buffer to each sample. Rotate the samples for 15 minutes at room temperature, then centum at 0.4 RCF for one minute.
Now save the supernatant in a two mil orang tube. Then add 250 microliters of evolution buffer to each sample. Once again, vortex them all by hand for a few seconds.
Then vortex the samples for 15 minutes after vortexing for 15 minutes, spin the tubes in the centrifuge. Add 16 RCF for four minutes and save the snat in the same two mill oring tube. To digest the protein in our samples, first add 10 microliters of 0.5 molar A DTA 25 microliters of 0.8 molar twist HCL L at a pH of 6.5 and 2.5 microliters of 10 milligrams per milli proteinase K to each chromatin immunoprecipitation sample.
Next to each input control add 15 microliters of lysis buffer and 2.5 microliters of 10 milligrams per ml. Proteinase K incubate both input and chromatin immunoprecipitation samples in 52 degrees Celsius for at least three hours. Now that the three hour incubation is over, we can go on and begin phenyl chloro Fromm extraction.
After the three hour incubation, we add 500 microliters of phenyl chloro fromm to each sample. This is done in the hood due to the fact that phenyl chloroform is an organic chemical that is both harmful if it comes in contact with the skin and also one inhaled. Next, we vortex all samples for several seconds and centrifuge them at 13 RCF for five minutes after centrifugation.
There should be two phases present within the tubes. Since we're interested in the contents of the top phase, we'll take out the top phase and put it into a two mil micro centage tube. Next, a mixture of two microliters of glycogen and 50 microliters of three molar sodium acetate is added to each sample along with 1.375 mils of a hundred percent ethanol.
All samples are vortex vigorously and placed in minus 80 degrees Celsius overnight. After the overnight incubation, the samples are placed on ice to thaw. They are dense centrifuge at 15 RCF for 10 minutes at four degrees Celsius.
After centrifugation, carefully remove the SUP natin without disturbing the pellet at the bottom of the tube. Next, add one mil of cold, 75%ethanol to each sample and invert them all four to six times. The tubes are then centrif again at 18 RCF for five minutes at four degrees Celsius.
The supernatant is once again removed and the pellets are allowed to air dry. Once the pellets are dry, they're dissolved in 50 microliters of four millimolar trace HCL of PH eight, and are stored at minus 80 degrees Celsius. Until further use, well, I've just shown you how to do chromatin immuno precipitations on frozen human postmortem brain tissue.
And remember, when doing this procedure, it is important to take all the necessary precautions when dealing with human brain tissue, chromatin, immunoprecipitation assays can be used to add an additional layer of understanding of gene expression within both the normal and diseased human brain. So that's it. Thanks for watching and good luck with your experiments.
